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Home , Apophyllum, Cleomaceae, Codonocarpus, Crateva, Emblingia, Forchhammeria

Systematic Botany (2004), 29(3): pp. 654–669 ᭧ Copyright 2004 by the American Society of Taxonomists

Molecular Phylogenetics of Core , Placement of Orphan Genera , , , and Character Evolution

JOCELYN C. HALL,1,2 HUGH H. ILTIS,andKENNETH J. SYTSMA Department of Botany, University of Wisconsin, Madison, Wisconsin 53706; 1Current Address: Arnold Arboretum, Dept OEB, Harvard University, 22 Divinity Ave., Cambridge, MA 02138; 2Author for Correspondence (email: [email protected])

Communicating Editor: Alan Meerow

ABSTRACT. Many genera previously placed in the traditionally circumscribed are either unrelated or, more commonly, isolated lineages in the order Brassicales. This study examines the relationships of three of these orphan genera, Emblingia, Forchhammeria,andTirania, in the context of a focused analysis of the core Brassicales. In order to assess rela- tionships of these genera, analyses were conducted across Brassicales using chloroplast rbcL, ndhF, and matK sequence in- formation. Both maximum parsimony and maximum likelihood analyses place all three genera in the well-supported core Brassicales (, Capparaceae, , , Pentadiplandraceae, , and Tovariaceae). The Asiatic Tirania and New World tropical Forchhammeria are closely related to two small families, the pan-temperate Resedaceae and the Australian Gyrostemonaceae. These analyses also indicate a novel placement of Emblingia as sister to all remaining members of core Brassicales. Although there is strong support for the relationships among most of these taxa, relationships of Pentadiplandraceae and Tovariaceae are weakly resolved. Thus, the core Brassicales is a biogeographically dispersed lineage that is comprised of many small and morphologically distinct clades plus the large crown group Brassi- caceae s. lat. Patterns of morphological evolution appear complex, especially in floral merosity and carpel and number. Likewise, the evolution of breeding systems within this lineage involves recurrent shifts towards monoecy or dioecy, and possible reversals to bisexuality. Further sampling of Capparaceae tribe Stixeae is critical for any taxonomic recommendation of familial status for these orphan genera.

The pantropical family Capparaceae Jussieu, with et al. 1999). Two prime examples include up to 45 genera and 900 , has long presented and , monotypic genera placed in Capparaceae enormous difficulties related to monophyly and gener- s. lat. that have been elevated to familial status and are ic placements. Recent molecular and morphological seemingly isolated members of Brassicales (Rodman et phylogenetic analyses of Capparaceae s. lat. and close- al. 1996, 1998). ly related Brassicaceae in the order Brassicales indicate Recent studies indicate two genera placed in Cap- that Capparaceae s. lat. as traditionally circumscribed paraceae s. lat., Emblingia and Forchhammeria, represent are paraphyletic (Rodman et al. 1993, 1996, 1998; Judd genera that are closely related to, but not part of, Cap- et al. 1994; Hall et al. 2002; Capparaceae s. lat. indicates paraceae s. lat. Emblingia is an enigmatic monotypic traditional, paraphyletic familial delimitation). Cap- that was placed in Capparaceae (Pax and Hoff- paraceae s. lat. comprise two monophyletic groups cor- man 1936), but has been recently aligned with Sapin- responding to the two major subfamilies, Cleomoideae daceae (Leins in Erdtman et al. 1969; Thorne 1992), and Capparoideae, with the Cleomoideae more closely (Melville and Metcalfe in Erdtman et al. related to Brassicaceae than to Capparoideae (Hall et 1969), or (Erdtman in Erdtman et al. al. 2002). These three taxa have been either combined 1969; Cronquist 1981). Based only on rbcL sequences, into one family, Brassicaceae s. lat. (Judd et al. 1994; Emblingia was tentatively linked with Resedaceae in APG 1998, 2003), or elevated to three separate families, the core Brassicales (Chandler and Bayer 2000), a clade Capparaceae s. str. (equivalent to Capparoideae), Cleo- in the order comprising Brassicaceae, Capparaceae, maceae (Airy Shaw 1965), and Brassicaceae (Hall et al. Cleomaceae, Gyrostemonaceae, Pentadiplandraceae, 2002). In this paper, we use the later classification Resedaceae, and Tovariaceae (Rodman et al. 1993, based on both morphological and molecular grounds 1996, 1998; Hall et al. 2002). Chloroplast sequences of (Hall et al. 2002). In addition to challenges of mono- ndhF and trnL-trnF placed the largely Central Ameri- phyly of Capparaceae s. lat. and issues of familial rank, can Forchhammeria within the core Brassicales and as there have been numerous challenges based on both sister to either Resedaceae or Gyrostemonaceae (Hall morphological and molecular evidence for placement et al. 2002). The ten species of Forchhammeria are rela- of genera within Capparaceae s. lat. (Table 1). Al- tively nondescript, dioecious that have been as- though some of these genera are now removed from signed to numerous families by a variety of taxono- Brassicales entirely (e.g., Calyptrotheca and Physena to mists, and their placement in Capparaceae s. lat. has ), most questionable genera remain in often been regarded as provisional (Hansen 1977). Pax Brassicales but outside Capparaceae or Cleomaceae and Hoffmann (1936) placed Forchhammeria in the tribe (e.g., Pentadiplandra, Villiers 1973; Setchellanthus, Karol Stixeae with Neothorelia, ,andTirania. Based on

654 2004] HALL ET AL.: PHYLOGENETICS OF CORE BRASSICALES 655 Ordinal (APG 1998) Classification Brassicales Brassicales Caryophyllales Brassicales Brassicales Caryophyllales emonaceae, emonaceae, In Erdtman et al. (1969), each of the four 1 classification Current familial this study ed by Kubitzki 2003f) 1997) this study Bayer 2000) lace 2001) Setchellanthaceae (Iltis 1999) Brassicales Physenaceae (Morton, Karol, and Chase Koeberliniaceae (Rodman et al. 1996) Brassicales Near Resedaceae and Gyrost Emblingiaceae (APG 1998; Chandler and relationships ndaceae (Erdtman et al. apindaceae, Oleaceae, Mal- Other suggested ) 1 phorbiaceae, S Dickison and Miller 1993) vaceae (Hansen 1977) affinity to Sapi 1969 Capparaceae s. str. and Cleomaceae) by Pax and Hoffmann (1936) and Brandegee (1909). ϭ Emblingia. Capparaceae s. lat. ( Subfamily (Tribe) of nieae) Pax and Hoffmann (1936) Buhsioideae Resedaceae (Hutchinson 1973; not accept- onomic placement of Liebm. Capparoideae (Stixeae) Eu Baill Pentadiplandroideae Near Celestraceae (Hutchinson 1973) Pentadiplandraceae (Villiers 1973) Brassicales Brandegee N/A Gilg. Calyptrothecoideae Portulacaceae (Nyananyo 1986) Near Didiereaceae (Applequist and Wal- Zucc. Capparoideae (Koeberli- Gagnep. Capparoideae (Stixeae) Uncertain (Kers 2003) F. Muell. Emblingioideae Polygalaceae, affinity with Goodeniaceae, or Chiovenda 1. Problematic genera placed in Norinha ex Thouars Capparoideae (Stixeae) Passifloraceae or Flacourtiaceae (reviewed in Pierre Capparoideae (Stixeae) Near Resedaceae and Gyrost Benge Buhsioideae Uncertain Genus, traditionally Lour. Capparoideae (Stixeae) Uncertain (Kers 2003) of Capparaceae s. lat. ABLE T Stefanina Stixis Tirania Setchellanthus Physena Neothorelia Pentadiplandra Forchhammeria Buhsia Calyptrotheca Emblingia authors hypothesizes a different tax 656 SYSTEMATIC BOTANY [Volume 29

FIG. 1. Distributions of ‘orphan’ genera of Capparaceae s. lat. and their putative relatives: Emblingia, Forchhammeria, Gyros- temonaceae, Pentadiplandraceae, Resedaceae, Tirania, and Tovariaceae. differences in number and number of wood anatomy (Carlquist 1985; Thorne 1992), this from Capparaceae s. lat., Kers (2003) excluded all coarse is distinct in its typically octmerous flow- members of Stixeae from his recent generic level treat- ers with plurilocular ovaries with axile placentation ment of Capparaceae s. lat. Further phylogenetic stud- and developed endosperm (Cronquist 1981; Appel and ies within the core Brassicales are thus warranted, as Bayer 2003), a distinction supported by molecular data only one species of Forchhammeria was sampled and (Rodman et al. 1993, 1996, 1998). Gyrostemonaceae putative relatives of Forchhammeria,suchasTirania,in and Resedaceae represent the remaining members of the tribe Stixeae have not been sampled. the core Brassicales. Gyrostemonaceae comprise five Perhaps more importantly from evolutionary and genera and 18 species of dioecious shrubs. Resedaceae biogeographical perspectives, the isolated genera and comprise approximately six genera and 70–75 species small families of the core Brassicales exhibit remark- of herbs or small shrubs with variable breeding sys- able variation in habit (annual, perennial herb or tems. Examination of habit, morphological characters, shrub, climbing shrub), floral structure (carpel, locule, and breeding systems of these problematic genera us- , and perianth number), breeding systems (bi- ing an explicit phylogenetic framework should provide sexual, monoecious, dioecious, polygamous), and dis- insight into their evolution and biogeography. junct areas of endemism (Fig. 1). Emblingia is a small Members of the core Brassicales, other than Bras- shrub possessing five , two , a gynophore, sicaceae s. str., Cleomaceae, and Capparaceae s. str., and a multi-carpellate with replum. The unique exhibit widely disparate biogeographical distributions of Emblingia has short colpi with rounded ends (Fig. 1), although many are found or centered in drier and exine that thickens close to the apertures such that habitats (e.g., Mediterranean biome). The distribution the apertural areas bulge out (Erdtman et al. 1969; Ku- of Forchhammeria is limited to Mexico, Central America, bitzki 2003a). The in Forchhammeria flowers is and the West Indies, where seven of the ten species are bicarpellate and bilocular with each locule usually con- rare and local, mostly in either dry or seasonally dry taining two seeds early in development. However, only areas (Hansen 1977). Other members of tribe Stixeae one of the seeds usually develops, giving the appear- putatively related to Forchhammeria are restricted to ance of being unilocular (Hansen 1977). The petals of southeast (e.g., Tirania). Like Emblingia, Gyroste- Tirania have a ligulate rim similar to Pentadiplandra monaceae are endemic to Australia where most species (Kers 2003). Pentadiplandra is a monoecious, climbing are fire-opportunists or of disturbed areas shrub possessing pentamerous flowers with more than (George 1982). The monotypic Pentadiplandraceae and three carpels and locules. Although some authors in- Tovariaceae (questionably ditypic) are restricted to clude Tovaria in Capparaceae s. lat. based on and tropical Africa and America, respectively. Whereas To - 2004] HALL ET AL.: PHYLOGENETICS OF CORE BRASSICALES 657 varia is found in cloud forests (Appel and Bayer 2003), subgenera recognized by Hansen (1977). The monotypic Tirania, an additional member of the tribe Stixeae, and Emblingia were in- Pentadiplandra grows in savannas, rain forests, and sec- cluded in the ndhF and rbcL data sets. Outside of the core Brassi- ondary growth (Ronse Decraene 2002; Bayer and Ap- cales, sequences were obtained for six additional families (seven pel 2003a). Only Resedaceae have a widespread dis- in the rbcL analysis that also samples Salvadoraceae), with one tribution restricted to the more arid parts of the North- species representing each family. Bretschneideraceae, Koeberlini- aceae, and Setchellanthaceae are monotypic and, Caricaceae, Mor- ern Hemisphere, mostly in the Old World but with a ingaceae, and Tropaeolaceae are each monophyletic based on mo- few species in southwestern United States and north- lecular data (Gadek et al. 1992; Andersson and Andersson 2000; ern Mexico. A more detailed phylogenetic analysis of Olson 2002a, b). Tropaeolum and Bretschneidera have been identified as sister to the remaining Brassicales (Rodman et al. 1993, 1994, the core Brassicales may provide insights into under- 1996, 1998) and were designated as a monophyletic outgroup. A standing their peculiar biogeographical patterns. suite of floral and embryological characters supports Tropaeolum Presented here is a phylogenetic analysis of the and Bretschneidera as a monophyletic group (Ronse Decraene et al. Brassicales with emphasis on the core Brassicales us- 2002). For the majority of the 38 taxa sampled, all three were obtained from the same DNAs or species (Appendix 1). In rare ing chloroplast sequence information from three cod- cases, the sequence was obtained from closely related species (e.g., ing regions. ndhF (Alverson et al. 1999; Hall et al. 2002; Capsella in matK). Sytsma et al. 2002), rbcL (Alverson et al. 1998; Conti et Extraction, Amplification, and Sequencing. Total genomic DNA al. 1996, 1997, 2002; Rodman et al. 1996, 1998; Sytsma was extracted from fresh, frozen, silica-dried or herbarium sam- ples using a modified CTAB method (Doyle and Doyle 1987; Smith et al. 2002), and matK (Koch et al. 2001; Sytsma et al. et al. 1991) or Dneasy Plant Mini Kits (Qiagen, Valencia, Califor- 2003; Wilson et al. 2001) have been shown to be infor- nia, USA). Standard polymerase chain reaction (PCR) and cycle mative at the infrafamilial level in Brassicales and re- sequencing techniques were used to amplify and sequence double stranded DNA (e.g., Hall et al. 2002). The 3Ј end of the ndhF lated orders Malvales, Sapindales, and Myrtales. In or- was amplified using forward primer 972F and reverse primer der to address questions on relationships among fam- 2110R from Olmstead et al. (1993) or slightly modified based on ilies of the core Brassicales and the placement of Em- Arabidopsis sequences (Hall et al. 2002). Four primers were used to blingia, Forchhammeria,andTirania, we added sequence both strands of the ndhF gene: 972F, 1603R, 1318F and 2110R. The rbcL gene was either amplified in one reaction using approximately 60 new sequences to the Rodman et al. 5Ј and 3Ј primers or in two reactions using two primer pairs, 5Ј/ (1996, 1998) rbcL, Koch et al. (2001) matK, and our own 674R and 523F/3Ј (see Conti et al. 1997 for details). Four sequenc- (Hall et al. 2002) ndhF data sets. With increased sam- ing reactions were conducted using primers 5Ј,3Ј, 523F, 674R, al- lowing for verification of both strands. Occasionally, other primers pling of taxa within the core Brassicales and combined (1020F, 1088R, 895R) were used for sequence verification. The matK sequence information from three gene regions, we region was amplified using trnK-710F and matK-1495R primers should be able to improve phylogenetic resolution. (Koch et al. 2001). A combination of primers was used to sequence This study has three major goals: (1) determine rela- this region: 1F, 16F, 495F, 495R, 1010R, 1010F, 1088R, 1412F, 1412R, and 1495R (Koch et al. 2001). In almost every instance, both tionships of lineages within the core Brassicales, spe- strands of each of the three genes were obtained. In the few ex- cifically relationships among Gyrostemonaceae, Rese- ceptions where this was not possible, manual proofreading of se- daceae, Tovariaceae, and Pentadiplandraceae; (2) deter- quences was done in order to detect any misreads. mine the placement of three of the unusual genera of Sequences were aligned using Sequencher v.3.0 (Gene Codes Corporation, Ann Arbor, Michigan, USA) and alignment was fur- Capparaceae s. lat., Forchhammeria, Tirania,andEmblin- ther refined using Se-Al v.2.0a6 (Rambaut 2001) or MacClade gia, within the core Brassicales; and (3) elucidate pat- v.4.05 (Maddison and Maddison 2002). All regions were codon terns, based on these molecular phylogenetic esti- aligned using the known Arabidopsis sequences. Whenever possi- ble, rbcL sequences were also checked for an extension past the mates, of evolution in floral morphology and breeding typical termination point, which has been demonstrated to be systems within core Brassicales. characteristic of core Brassicales and near relatives (Rodman et al. 1994, 1996; Karol et al. 1999). Previous phylogenetic studies have noted indel events both in ndhF (Hall et al. 2002; Sytsma et al. MATERIALS AND METHODS 2002) and matK (Koch et al. 2001) that can be phylogenetically Taxon Sampling. We sampled widely within the Brassicales informative. Indels that were potentially parsimony informative (sensu Rodman et al. 1998; Appendix 1) and covered 13 of the 16 (e.g., shared by two or more but not all taxa) were scored and families in the order excluding Limnanthaceae, Setchellanthaceae, added to the end of the data set as presence/absence characters and Akaniaceae (which has been recently combined with Bret- following the guidelines of Baum et al. (1994). schneidera into one family [Bayer and Appel 2003b]). Rodman et Phylogenetic Analyses. Variation in DNA sequences was used al. (1998) did not sample Emblingia so this taxon was not included to reconstruct phylogenetic relationships using maximum parsi- as a member of the order until APG (1998) and Chandler and mony (MP) and maximum likelihood (ML) in PAUP* (v. 4.0b10; Bayer (2000) placed it there. Our sampling includes all families of Swofford 2002). All three data sets were analyzed individually and core Brassicales. Although Capparaceae, Cleomaceae, and Brassi- in combination. Phylogenetic data sets have been deposited in caceae represent the three largest clades of the core Brassicales, TreeBASE (study accession number S1000). To explore the possi- we sampled only four to five representatives from each family bility of multiple islands of most parsimonious trees, 1000 random because all three are well supported as monophyletic, and taxa addition replicates with Multrees (save multiple trees) holding five selected represent both the smaller, early diverging lineages and trees at each step and TBR branch swapping were used. All char- the larger subclades within each family (Hall et al. 2002). A single acters were equally weighted and treated as unordered (Fitch representative each from Pentadiplandraceae (monotypic) and To- 1971). In analyses of ndhF and matK, these data were analyzed variaceae (monotypic or questionably ditypic [Appel and Bayer with and without scored indels. In addition to standard measures 2003]) were available. Resedaceae and Gyrostemonaceae had two of fit of characters to the trees produced (i.e., consistency index, and three representatives, respectively. Five species of Forchham- retention index), the strength of support for individual branches meria were sampled (for both rbcL and matK), representing all three was estimated using the bootstrap (Felstenstein 1985). Bootstrap 658 SYSTEMATIC BOTANY [Volume 29 analyses used 1000 replicates (simple addition, saving up to 1000 Bayer 2003; Bayer and Appel 2003a-c; Kubitzki 2003a–g). Charac- trees per replicate, TBR branch swapping, multrees, holding 1 tree ter states were scored for all species present in the combined anal- per step). yses of 35 taxa and then mapped onto the maximum likelihood Fifty-six maximum likelihood models were explored in individ- tree from that analysis using the TRACE CHARACTERS function ual and combined data using Modeltest v.3.06 (Posada and Cran- in MacClade, which explores both Deltran and Acctran resolving dall 1998). To establish which model of DNA substitution best fits options. The topologies of the maximum likelihood and maximum the data, this program compares these ML models in a hierarchical parsimony trees differed only with respect to the position of Pen- testing framework by calculating the likelihood ratio statistic be- tadiplandra, so both positions were explored when mapping char- tween different models. Likelihood ratio tests and their associated acters. In rare instances, taxa were polymorphic but were scored p-value compare alternative models of sequence evolution and im- as having one state (see discussion for details). provement in fit with increasing model complexity. A heuristic ML search with TBR branch swapping was then conducted using pa- ESULTS rameters determined for the best model of sequence evolution. R Combined analyses were conducted with two different sam- pling schemes. The first included only the 31 taxa for which se- Phylogenetic Relationships. Although there are quence information existed for all three genes. The one exception minor differences between individual and combined is Aethionema, which has a critical relationship of being sister to analyses, there was very strong congruence in topol- all other Brassicaceae s. str. (Galloway et al. 1998). Aethionema was ogies (Figs. 2, 3). All analyses support the core Brass- represented as a composite terminal taxon based on Aethionema saxatile and Aethionema grandiflora. This approach was justified by icales as a monophyletic clade. Koeberlinia and Batis the strong sister relationship of the two taxa species with the ndhF form a sister group to the core Brassicales (with the analyses. However, this reduced 31 taxon sample excluded some exception in rbcL analysis) and Caricaceae and Mor- critical taxa, specifically Emblingia, one species of Forchhammeria, ingaceae are sister to these two clades. Within the core and Tirania, for which we were not able to obtain sequence data for matK. To evaluate the relationships of these taxa, we conducted Brassicales, all families and genera with more than one parsimony and maximum likelihood analyses on an expanded species sampled (Capparaceae, Cleomaceae, Gyroste- combined analysis with 35 taxa to include all taxa for which there monaceae, Forchhammeria, and Resedaceae) were was missing data (Aethionema, Emblingia, Forchhammeria pallida, and Tirania). monophyletic. Brassicaceae s. lat. form a monophyletic The incongruence length difference (ILD) test (Farris et al. 1994, lineage with Cleomaceae and Brassicaceae s. str. as sis- 1995), implemented in PAUP* as the partition homogeneity test, ter groups. Forchhammeria is consistently sister to Re- was conducted to measure conflict between the three data sets. sedaceae, with Gyrostemonaceae sister to these two Although aspects of the ILD test have been criticized (Yoder et al. 2001; Barker and Lutzoni 2002; Darlu and Lecointre 2002), Hipp clades, and is clearly outside the Capparaceae. Al- et al. (2004) have demonstrated that some of these criticisms are though Tirania is most closely related to Resedaceae invalid and argue that the ILD has merit as a first estimate of and Forchhammeria, the precise relationships among combinability of data sets. A three-way test on parsimony-infor- mative characters of 1000 replicates (simple addition, TBR branch these three clades are unresolved. For ease of discus- swapping) was performed, saving only 1000 trees per replicate. sion, we refer to the lineage containing - Pair-wise ILD tests were also conducted using the same search aceae, Resedaceae, Forchhammeria,andTirania as the strategy above on each pair of data sets. GRFT clade. Emblingia is sister to all other core Brass- Alternative a priori taxonomic hypotheses were explored by en- forcing topological constraints, using 100 random addition repli- icales in ndhF and rbcL analyses (not sampled for cates and calculating the number of additional steps required. Al- matK). Relationships of Tovariaceae and Pentadiplan- ternative relationships suggested by individual, but not the com- draceae within the core Brassicales vary among all bined, analyses were also examined in similar fashion. As pres- ently no parsimony based statistical test of such tree differences analyses with little support for alternative topologies. (a priori vs. a posteriori derived trees) are generally accepted rbcL Analysis. In parsimony analyses of the rbcL (Goldman et al. 2000), we implemented ML based tests of tree sequences of 35 taxa, 48 most parsimonious trees of differences using the Shimodaira and Hasegawa (1999) parametric length 817 on a single island were recovered (Table 2). test (SH test). Following guidelines for tree selection in Buckley et al. (2001, 2002), a tree set including a number of the a priori tax- The aligned length of the region is 1479 of which 242 onomic hypotheses previously explored plus a posteriori ML trees (16.4%) characters were parsimony informative (Table from individual and combined analyses were evaluated with the 2) and no indels were introduced during alignment. SH test in PAUP* with RELL optimization using ML settings for Only partial sequences were obtained for Crateva pal- the combined data set. Character Evolution. Patterns of morphological evolution were meri, dodecandra,andWislizenia refracta (with assessed in MacClade by mapping characters of interest onto the sequence lengths of 864, 787, and 851, respectively). most parsimonious trees obtained from the combined parsimony There are 14.2% cells missing in this data set. All Gy- analysis. The following six characters were evaluated: 1. habit (a. annual herb, b. perennial herb, c. woody; unordered), 2. carpel rostemonaceae share the same c-terminal amino acid number (a. two, b. three, c. Ͼ3; both ordered and unordered), 3. sequence inferred from nucleotide sequences as pre- locule number (a. two, b. three, c. Ͼ3; both ordered and unor- viously reported (Karol et al. 1998). The inferred ami- dered), 4. number (a. Ͻfour, b. four, c. five, d. six, e. irregular no acid at positions 1426–1428 in the rbcL sequences of [unevenly lobed]; unordered), 5. number (a. Ͻfour,b.four, c. five, d. six, e. irregular [unevenly lobed], f. absent; unordered), Emblingia, Forchhammeria watsonii, F. sessilifolia, F.sp. and 6. breeding system (a. hermaphroditic, b. monoecious, c. di- nov., and Tirania is aspartic acid (rather than a stop oecious, d. polygamous; unordered). Morphological data were de- codon that is typical of many dicots), a synapomorphy termined using field studies, herbarium sheets, and literature for the core Brassicales (Rodman et al. 1994, 1996; Ka- (Hansen 1977; Cronquist 1981; George 1982, 2003; Rodman et al. 1991; Hufford 1996; Olson 2002a, b; Ronse Decraene 2002; Ronse rol et al. 1999). Unfortunately, for most of these taxa Decraene et al. 1998, 2002; Appel and Al-Shehbaz 2003; Appel and our sequences ended at this codon position so we are 2004] HALL ET AL.: PHYLOGENETICS OF CORE BRASSICALES 659

FIG. 2. Phylogram results of parsimony analyses for individual and combined data sets, showing only key relationships within the core Brassicales. Bootstrap values are above branches and for Forchhammeria, Resedaceae, and Gyrostemonaceae the values indicate monophyly for those clades. (A) Consensus of the 48 trees based on analysis of 35 sequences of rbcL. (B) The most parsimonious tree in analyses of 35 ndhF sequences. (C) Consensus of the matK data set of 31 taxa, excluding Tirania and Emblingia. (D) Eighty percent bootstrap consensus of expanded combined analysis (35 taxon sample) of rbcL, ndhF and matK including taxa with missing sequences for matK (Tirania and Emblingia). not able to examine the extent of the ‘tail.’ The strict among site rate heterogeneity is modeled by allowing consensus of the core Brassicales is shown with boot- some sites to be invariant (I) while the rest have rates strap values indicated (Fig. 2A). There is low support drawn from a discrete approximation to a gamma dis- (bootstrap 55%) for the monophyly of the core Brass- tribution with a single shape parameter, alpha (G; Ta- icales, including Emblingia, and for relationships of ble 2). The heuristic search converged on a single tree Pentadiplandra, Tovaria,andEmblingia within the core (ln L ϭϪ 6678.34464; tree not shown) that is nearly Brassicales (all Ͻ50% bootstrap). Emblingia is sister to identical in topology to the consensus of the parsi- all other core Brassicales whereas Tovaria and Pentadi- mony analysis. In the ML tree, Tirania is sister to Re- plandra are part of the GRFT clade. Forchhammeria and sedaceae and these two clades are sister to Forchham- Tirania are strongly supported as closely related to Re- meria. Also, within Cleomaceae, pilosa and C. sedaceae (bootstrap 98%) with the position of Tirania viridiflora are sister species whereas in the MP topology unresolved. Gyrostemonaceae is weakly supported as C. pilosa is sister to . The overall topology sister to Forchhammeria, Tirania, and Resedaceae (boot- of the ML tree is similar to analysis of rbcL by Karol strap 61%). Among other members of Brassicales, there et al. (1999), with the exception of the sister relation- is more than one most parsimonious relationship hy- ship of Tovaria and Pentadiplandra recovered in Karol et pothesized for Koeberlinia. al. (1999). Hierarchical likelihood ratio tests indicated that the ndhF Analysis. One most parsimonious tree best model of substitution for the rbcL data is TrN ϩ (length 1283) was recovered in parsimony analysis of I ϩ G, which allows one substitution rate for transver- 35 ndhF sequences (Fig. 2B). Six indels in lengths of 3, sions, two substitutions rates for transitions, and 6 or 9 base pairs were introduced during sequence 660 SYSTEMATIC BOTANY [Volume 29

FIG. 3. The maximum likelihood tree of the 35 taxa combined rbcL, ndhF, and matK analysis including Tirania and Emblingia under the TVM ϩ I ϩ G model of DNA substitution. The topology of this tree is almost identical to the maximum parsimony analyses with the exception of position of Pentadiplandra. Branch lengths are proportional to the number of changes along each branch. GRFT clade in shaded box. alignment. The entire aligned data set is 1097 base covered in the ndhF analyses differ from rbcL: (1) a pairs in length and 7.74% cells are scored as missing sister relationship of Maerua and Apophyllum (Cappar- (Table 2). Aethionema saxatile is missing approximately aceae; relationships not shown in figure), (2) Tovaria as one half of the sequence. Excluding or including the sister to Capparaceae, Cleomaceae plus Brassicaceae, scored indels even up to weight of 10 had no effect on and (3) Tirania as sister to a well supported (bootstrap resulting topology and only minor effect on relative 85%) clade of Resedaceae plus Forchhammeria. Emblin- bootstrap values. The partition homogeneity test in- gia is supported (bootstrap 87%) as sister to remaining dicates rbcL and ndhF have similar phylogenetic struc- core Brassicales. The heuristic search under likelihood ture (P ϭ 0.193). The following three relationships re- settings resulted in a single tree (ln L ϭϪ 8046.93736; 2004] HALL ET AL.: PHYLOGENETICS OF CORE BRASSICALES 661

TABLE 2. Characteristics of the three data sets and parsimony and maximum likelihood analyses. 1 Analyses excluding indels. 2 Model selected using hierarchical likelihood ratio tests in Modeltest (Posada and Crandall 1998).

rbcL ndhF1 matK1 Combined 1 Combined 2 Number of taxa 35 35 31 31 35 Aligned length 1479 1097 1566 4142 4142 Parsimony: Parsimony informative sites (%) 242 (16.4%) 342 (31.2%) 458 (29.2%) 988 (23.9%) 1031 (24.9%) MP trees (length) 48 (817) 1(1283) 6 (1582) 2 (3494) 2 (3660) CI (RI) 0.661 (0.704) 0.633 (0.726) 0.677 (0.760) 0.674 (0.741) 0.656 (0.736) Maximum likelihood: ML model2 TrN ϩ I ϩ G GTR ϩ G TVM ϩ G TVM ϩ I ϩ G TVM ϩ I ϩ G Likelihood score (-ln L) 6678.34464 8046.93736 10490.42846 24508.01206 25390.54641 Base frequencies A 0.2845 0.3416 0.3272 0.3143 0.3147 C 0.1886 0.1291 0.1463 0.1571 0.1577 G 0.2397 0.1322 0.1453 0.1745 0.1723 T 0.2872 0.3972 0.3812 0.3540 0.3553 Substitution rates A-C 1.0000 1.4130 1.5828 1.4784 1.4329 A-G 1.9635 2.1857 2.1531 2.1438 2.1309 A-T 1.0000 0.2872 0.3107 0.4356 0.4261 C-G 1.0000 2.1703 1.6964 1.4891 1.4609 C-T 2.6116 1.8780 2.1531 2.1438 2.1309 G-T 1.0000 1.0000 1.0000 1.0000 1.0000 Proportion invariant sites (I) 0.5418 0 0 0.2603 0.2906 Gamma distribution shape parameter (G) 0.7781 0.6765 1.1023 0.9474 1.0425

tree not shown) that is identical to the parsimony tree, structure of matK with rbcL (P ϭ 0.684) and with ndhF except the relationship of Tovaria is unresolved (there (P ϭ 0.934). The best model of DNA substitution for is a hard polytomy). The best model of substitution for the matK data is the transversional model TVM ϩ G the ndhF data set is GTR ϩ G that allows for six dif- in which there are four different transversion rates, one ferent substitution rates for all nucleotide pairs and transition rate, and rate heterogeneity is approximated among site rate heterogeneity is approximated by a using the gamma distribution. The topology of the sin- gamma distribution (Table 2). gle tree resulting from maximum likelihood analyses matK Analysis. Six most parsimonious trees of of the matK data shows a topology (ln L ϭϪ length 1582 resulted from analyses of 31 matK se- 10490.42846; tree not shown) in which Pentadiplandra is quences (Fig. 2C; Table 2). The aligned length of the sister to the Capparaceae, Cleomaceae, and Brassica- data set was 1566 base pairs with five indel events in- ceae with Tovaria sister to these two clades. troduced. For some taxa we were not able to obtain Combined Analysis. The three-way partition ho- complete sequences and 5.44% of the data set is scored mogeneity test, conducted on the smaller 31 taxon as missing. Including or excluding the scored indels sample, indicated all three data sets have similar phy- altered topology and support of resulting trees. The logenetic structure (P ϭ 0.414). Parsimony analyses on inclusions of weighted indels dissolve the following taxa for which sequences were available for all three sister relationships: (1) Moringa and Carica, and (2) Ba- regions (with the exception of Aethionema treated as a tis and Koeberlinia. These relationships are well sup- single taxon) resulted in two most-parsimonious trees ported in other analyses (Figs. 2, 3; Rodman et al. (length 3494; tree not shown), which differ only in the 1993, 1998; Olson, 2002a, b), indicating the indels in relationships among species of Forchhammeria. The to- matK are homoplasious and justifiably excluded from pology is highly congruent with all individual analy- analyses. The relationships within the core Brassicales ses, and bootstrap support for all branches is in- and the placement of Forchhammeria are not altered creased. The combined analysis with 31 taxa suggests with the inclusion of indels. The resulting topology of relationships of Tovaria and Pentadiplandra not indicated the matK parsimony analysis is highly congruent with in the individual analyses. Tovaria is sister to all other both ndhF and rbcL analyses (Figs. 2, 3). Tovaria and core Brassicales (bootstrap 71%) whereas Pentadiplan- Pentadiplandra are both unresolved by matK analyses. dra is sister to Capparaceae, Cleomaceae, and Brassi- The ILD test indicates there is similar phylogenetic caceae (bootstrap 60%). In the expanded combined 662 SYSTEMATIC BOTANY [Volume 29 analysis with 35 taxa, two trees resulted in which the Tirania, and Tovariaceae. The GRFT clade of Gyroste- overall topology is almost identical to the 31 taxon monaceae, Resedaceae, Forchhammeria,andTirania is a sample tree (Fig. 2D). The two most parsimonious novel lineage established here, with problematic affin- trees differ with respect to Pentadiplandra. One topol- ities to Pentadiplandra and Tovaria. Ronse Decraene ogy is identical to the 31 taxon sample tree topology (2002) suggested that racemose inflorescences, (andro) (e.g., sister to Brassicaceae s. lat.), whereas the other gynophore, extrastaminal nectary, tendency to dissym- topology indicates Pentadiplandra is sister to the GRFT metry, reduced stipules, and imbricate sepals and pet- clade. Tirania is placed in a polytomy with Resedaceae als unite the core Brassicales. Other characters that are and Forchhammeria,andEmblingia is sister to remaining common, but not necessarily synapomorphies, include core Brassicales (Fig. 2D). Relationships of Tovaria and campylotropous seed orientation (Hufford 1996) with Pentadiplandra are unresolved, although there is weak the exception of Forchhammeria (Hansen 1977), anom- support (bootstrap 60%) for Tovaria as sister to all core ocytic stomates, and curved embryos (also seen in Koe- Brassicales other than Emblingia. berlinia). The best model of DNA substitution for the com- Emblingia is Sister to Remainder of Core bined data set is TVM ϩ I ϩ G (transversional model) Brassicales. The studies presented here suggest a in which there are four different transversion rates, one novel relationship of Emblingia, as sister to remaining transition rate, and among-site rate heterogeneity is core Brassicales. A previous molecular study using modeled by allowing some sites to be invariant while rbcL placed Emblingia in Brassicales as sister to Rese- the rest have rates drawn from a discrete approxima- daceae but with low support (bootstrap Ͻ50%; Chan- tion to a gamma distribution. That model of evolution dler and Bayer 2000). The lack of resolution in their is the same for both 31 and 35 taxa data sets (Table 2), study was likely the result of a very broad sampling and the 31 taxon tree is identical to the 35 taxon tree across many orders that had been suggested to house (Fig. 3). Tirania and Emblingia have the same relation- Emblingia (Table 1; Chandler and Bayer 2000), limited ships as the rbcL, ndhF, and combined parsimony anal- sampling within core Brassicales, and reliance solely yses. The precise placement of Tirania is unresolved by on rbcL sequences. Although Emblingia has many likelihood analyses. unique morphological features, there are some features in common with other members of core Brassicales: DISCUSSION androgynophore (Erdtman et al. 1969; Chandler and Data from more than 60 new rbcL, ndhF, and matK Bayer 2000), curved or reniform seeds; tricolporate pol- sequences, individually and in combination, resolve len; usually 4-merous (or 8); and zygomorphic many relationships within the core Brassicales and flowers. To our knowledge, no studies have been con- help place Emblingia, Forchhammeria,andTirania. Sev- ducted to examine the presence of mustard oils (glu- eral results confirm relationships within Brassicales cosinolates) in Emblingia. and core Brassicales indicated in previous analyses Positions of Pentadiplandra and Tovaria are not (Rodman et al. 1993, 1994, 1996, 1998; Hall et al. 2002) Resolved. Although there were 1031 parsimony in- but with increased support. Novel phylogenetic results formative characters in the combined 35 taxon analy- that emerge from these studies include: (1) Emblingia sis, some relationships within core Brassicales are still is sister to remaining core Brassicales, (2) Gyrostemon- not resolved. Every analysis suggested different rela- aceae, Resedaceae, Forchhammeria,andTirania form a tionships of Tovaria and Pentadiplandra, each with low well-supported clade within the core Brassicales, and statistical support (Figs. 2, 3). Since both families are (3) the relationships of Pentadiplandra and Tovaria with- monotypic (Tovaria questionably ditypic), increased in the core Brassicales remain unresolved. Mapping of sampling is not a viable approach for resolving these morphological traits onto phylogenetic trees indicates relationships. Both genera have been classified as Cap- that many of the characters used to classify these taxa paraceae in the broad sense by some taxonomists (Pax are prone to convergent evolution. Although many pat- and Hoffmann 1936; Carlquist 1985; Thorne 1992), al- terns of morphological evolution remain elusive, some though Hutchinson (1973) placed Pentadiplandra near insight is gained on the evolution of floral merosity the Celastraceae. Ronse Decraene (2002) suggested a and breeding systems. Two other orphan genera of strong affinity of Tovaria and Pentadiplandra based on Capparaceae s. lat., Stixis and Neothorelia, which have the presence of plesiomorphic characters within Brass- been placed in the tribe Stixeae, clearly need to be sam- icales such as stipulate , pentamery, diploste- pled. mony, and axile placentation. However, none of the Monophyly of the Core Brassicales. The core Brass- analyses presented here indicate a sister relationship icales are a strongly supported clade (bootstrap 100% of these two genera (but see Rodman et al. 1998; Karol in combined analyses) comprising Brassicaceae, Cap- et al. 1999). paraceae, Cleomaceae, Emblingiaceae, Forchhammeria, Forchhammeria and Tirania are Related to Gyros- Gyrostemonaceae, Pentadiplandraceae, Resedaceae, temonaceae and Resedaceae. The clade comprising 2004] HALL ET AL.: PHYLOGENETICS OF CORE BRASSICALES 663

Gyrostemonaceae, Resedaceae, Forchhammeria,andTir- morphy for the GRFT clade, members of the clade have ania (GRFT clade) is moderately (bootstrap 61% in some morphological similarities or trends. Gyroste- rbcL) to strongly supported (bootstrap 94–99% in other monaceae, Forchhammeria,andOchradenus (Resedaceae, analyses). Gyrostemonaceae, here represented by three unsampled in these studies) all have uniseriate peri- species, is sister to all remaining members of the clade. anths, grow in arid habitats, and are anemophilous Despite the strong support for Forchhammeria, Tirania, (Hansen 1977; Hufford 1996). The flowers of Forchham- and Resedaceae as a clade, there is no consensus on meria, like Gyrostemonaceae, are much reduced with a how these three lineages are related. The combined fused calyx that splits in an irregular fashion. Gyros- data presented here indicate a sister relationship of temonaceae and Forchhammeria are dioecious with Forchhammeria and Resedaceae, a relationship further highly reduced flowers, presumably adapted for wind supported by a unique 9bp matK deletion. In a previ- pollination. However, many of these features may be ous analysis using only trnL-trnF data (Fig. 3 in Hall the result of convergent evolution due to dioecy and et al. 2002), Forchhammeria is placed sister to - arid habitat. Tirania and Resedaceae (excluding Ochra- ceae plus Gyrostemonaceae (bootstrap 83%). This denus) are the only members of the clade that have pet- analysis, however, was conducted with only one rep- als. Of some interest is that both genera sampled from resentative from each of the three lineages, and limited the tribe Stixeae of Capparaceae s. lat. (Pax and Hoff- sampling may be an issue. In order to evaluate the mann 1936), Forchhammeria and Tirania, are closely re- significance of these differences, the ML based Shi- lated to one another and Resedaceae. Members of the modaira-Hasegawa test was conducted on combined tribe Stixeae have two to multilocular ovaries, axile and the trnL-trnF data sets (trnL-trnF data set was ex- placentation, three- to five-merous perianth, and well panded to include all the same representatives of the developed stigmas (Pax and Hoffmann 1936; Kers GRFT clade, J. Hall unpublished data) under ML pa- 2003), which support their placement within the GRFT rameters determined by ModelTest (Posada and Cran- clade (Fig. 4). Precisely how Forchhammeria and Tirania dall 1998). Our a priori hypotheses included 15 topol- are related to one another is still unresolved based on ogies representing all possible relationships among the rbcL and ndhF sequence data (Figs. 2, 3). Additional four members of the GRFT clade. The combined data sampling of remaining Stixeae (i.e., Neothorelia and set rejects all topologies (P ϭ 0.001) except those in Stixis) is warranted based on the clear separation of which Gyrostemonaceae are sister to all other mem- these two genera from Capparaceae s. lat. and their bers of the GRFT clade (P ϭ 0.896–0.914). In contrast, close relationship. the trnL-trnF data set fails to reject any of the 15 to- Morphological Evolution. Mapping of character pologies (P ϭ 0.158–0.569). These analyses indicate states revealed few clear patterns of morphological that the sister relationship of Forchhammeria and Re- evolution within the Brassicales. Figure 4 maps breed- sedaceae suggested by combined matK, rbcL,andndhF ing systems and shows selected habit and floral char- is more likely than that indicated by the previous trnL- acters (mapping of these characters can be obtained trnF analysis, based on fewer taxa. from authors upon request). Moving Pentadiplandra as Although sampling is limited among some members sister to the GRFT clade does not change the recon- of the GRFT clade, all genera or families with more struction of any of the characters (typically the branch than one species sampled are strongly supported as is equivocal in either relationship) with the exception monophyletic with clear putative morphological syn- of breeding systems. Habit is the only morphological apomorphies. Resedaceae have a syncarpous gynoeci- character for which there are no equivocal branches in um that remains open at the distal end throughout de- the reconstruction. Woodiness is plesiomorphic in velopment (Cronquist 1981; Hufford 1996; Kubitzki Brassicales and, based on current sampling, the her- 2003f), although the family exhibits variation in carpel baceous habit has arisen four times: (1) Cleomaceae number. The massive embryos of Forchhammeria are plus Brassicaceae, (2) (Resedaceae), (3) Ter- pseudo-monocotyledonous (Hansen 1977), where only sonia (Gyrostemonaceae), and (4) Tropaeolum (Tropaeo- the outer cotyledon develops into a fat folded nutrient laceae). storage structure, completely enveloping the inner, un- Merosity of both the and perianth is very developed cotyledon and with the radicle highly re- labile within the Brassicales (Fig. 4). When designated duced (except Forchhammeria sp. nov.). Female flowers as unordered, the plesiomorphic state of locule and of Gyrostemonaceae are apopcarpic with carpels ad- carpel number is equivocal. When ordered, three car- nate to a central column that is often expanded at the pels are basal within the Brassicales, but the base of top (Cronquist 1981; Hufford 1996). In fact, the unusu- the core Brassicales is still equivocal. Within the core al syncarpy of Gyrostemonaceae appears to be asso- Brassicales, two carpels characterize the Capparaceae, ciated with the formation of a large, sterile region in Cleomaceae, and Brassicaceae clade in addition to the apex of flowers (Hufford 1996). Forchhammeria and Gyrostemon tepperi. Whether treated Despite the lack of a clear morphological synapo- as ordered or unordered, the primitive locule number 664 SYSTEMATIC BOTANY [Volume 29

FIG. 4. Optimization mapping of breeding systems on the maximum likelihood tree from the combined 35 taxa analysis. Character states for habit, sepal number, petal number, carpel number, and locule number are given. Wϭ woody perennial, P ϭ herbaceous perennial, H ϭ annual herbaceous, I ϭ irregular. for the core Brassicales is equivocal. Within the core bisexual (Fig. 4). Based on this sampling, dioecy Brassicales, two locules is characteristic of both Bras- evolved unambiguously from the hermaphroditic con- sicaceae s.str. and Forchhammeria. The evolution of peri- dition three times (Carica, Batis, Apophyllum), and mon- anth number is equally ambiguous, although merosity oecy or polygamy evolved twice (Crateva, Pentadiplan- characterizes a few clades. Within the core Brassicales, dra), also unambiguously. The evolution of dioecy all dioecious plants lack petals and all but one have within the GRFT clade is ambiguous, as a result of the irregular sepals. There is no clear pattern of sepal lack of resolution among Forchhammeria, Tirania,and number within the GRFT clade, except that in general Resedaceae. Alternative topologies of these three taxa they have irregular splitting sepals. The equivocal sta- were explored. If Forchhammeria is sister to Tirania plus tus of many of the characters at the base of the GRFT Resedaceae and Pentadiplandra is sister to the GRFT clade is due to the unresolved nature of Tirania. clade, then monoecy, dioecy, and hermaphroditism are The ancestral condition of breeding systems for the all possible ancestral conditions. In all other arrange- core Brassicales and Brassicales in the broad sense is ments of Forchhammeria, Tirania, and Resedaceae (re- 2004] HALL ET AL.: PHYLOGENETICS OF CORE BRASSICALES 665 gardless of placement of Pentadiplandra), the ancestral rived unisexual forms occur (Cronquist 1981; Kubitzki condition is hermaphroditism with two independent 2003f). Tovaria was scored with a merosity number of origins of dioecy. six, but merosity varies between six and eight (Ronse Biogeographical Relationships. Many members of Decraene 2002). the core Brassicales and the GRFT clade, in particular, Patterns of morphological evolution are limited by have disjunct distributions (Fig. 1). There is a strong taxon sampling in the analyses. Although Batis is Austral-Asian component in the clade as well as a pro- scored as dioecious, the family is ditypic, and the other pensity for Mediterranean habitats. The majority of species, not sampled in these analyses, is monoecious Gyrostemonaceae are endemic to southwestern Aus- (Bayer and Appel 2003b). Gyrostemon sp. (probably a tralia, occur in eastern Australia ( attenu- new species, see Rodman et al. 1994) was scored based atus) or throughout central Australia (Codonocarpus co- on generic characters, and the typical carpel number tinifolius) (George 1982, 2003). Resedaceae are primar- for most Gyrostemonaceae is six to eight (Hufford ily in arid regions of the Mediterranean, with outliers 1996). Gyrostemon tepperi typically has one to two car- from Oligomeris in South Africa and southwestern pels, which likely represents an evolutionary reduction (Kubitzki 2003f). Tirania is endemic to (Hufford 1996). Assuming that this assessment is cor- southern Vietnam (Hutchinson 1967; Kers 2003). Forch- rect and the reduced carpel number in G. tepperi is hammeria is distributed in Mexico, Central America, derived, an alternative scoring of G. tepperi was imple- and the West Indies (Hansen 1977). Hansen (1977) sug- mented with a high carpel number for the family. With gested that Forchhammeria was closely related to some this scoring, the GRFT clade (regardless of the position African genera of Capparaceae (Boscia and Maerua) of Pentadiplandra) has a plesiomorphic condition of high and that the current distribution of the genus was the carpel number with a reduction in Forchhammeria.In result of long distance dispersal. Given the relation- fact, the entire core Brassicales has a plesiomorphic ships presented here using chloroplast sequence data, character state of greater than three carpels with two the biogeographic history of Forchhammeria needs to be reductions: (1) Forchhammeria and (2) Brassicaceae plus examined instead relative to the temperate Resedaceae, Cleomaceae plus Capparaceae. Australian Gyrostemonaceae, and Asiatic Tirania. Floral dimorphism (dioecy, monoecy, or polygamy) These biogeographical relationships are not clearly re- evolved minimally eight times within Brassicales solved in the GRFT clade and become increasingly less based on current sampling (Fig. 4). This is likely an clear when the distributions of other core Brassicales underestimate of change in breeding systems because are considered: Pentadiplandra of tropical west Africa, there are many unsampled species in Capparaceae and Tovaria of tropical America, Emblingia of Australia, and Cleomaceae that are either monoecious or polygamous. the worldwide distributed Brassicaceae, Cleomaceae, Dioecy has evolved four to five times within Brassi- and Capparaceae. A preliminary molecular clock dat- cales: Carica papaya (Caricaceae), Batis maritima (Bata- ing of these lineages (Hall 2003) suggests that the core ceae), Apophyllum (Capparaceae), Gyrostemonaceae, Brassicales diversified during the late Cretaceous and and Forchhammeria. The transition to dioecy is from an early Tertiary, but more detailed analyses are needed hermaphroditic ancestor except for Forchhammeria and to understand biogeographical relationships and Gyrostemonaceae, where the ancestral state is equiv- events within the core Brassicales in the context of con- ocal. Because the sister relationship of Forchhammeria tinental separations and contacts. and Gyrostemonaceae is rejected by the Shimodaira- Floral Divergence and Convergence in Core Hasegawa test, two independent origins of dioecy Brassicales. One result of using a molecular phylo- from hermaphroditism are likely. The shifts to dioecy genetic framework to address patterns of morpholog- from hermaphroditism differ from the general pattern ical evolution in the Brassicales is the demonstration of (Renner and Ricklefs 1995), but are similar to the pat- the diversity and lability of floral merosity in the order. tern of breeding system shifts within monocots (Wei- Recurring or convergent shifts in sepal, petal, carpel, blen et al. 2000). Generally, hypotheses of evolutionary and locule number are common, especially within the pathways to dioecy include gynodioecious or monoe- core Brassicales (Fig. 4). In assigning character states cious intermediates (Charlesworth and Charlesworth to taxa, some of this diversity was necessarily simpli- 1978; Sytsma et al. 1991; Barrett 2002). The direct tran- fied from even greater complexity in floral evolution of sition between hermaphroditism and dioecy may be Brassicales. Resedaceae were scored as having greater spurious as transitions can be missed when looking at than three carpels (plesiomorphic condition), although phylogenetic reconstructions (Weiblen et al. 2000). the family varies in carpel number between two and Dioecy has been associated with many characteris- seven: Reseda has three to four whereas Oligomeris has tics in flowering plants including wind pollination, pe- four to five (Cronquist 1981; Kubitzki 2003f). Also, al- rennial growth, and fleshy (Givnish 1980; Ren- though flowers of Resedaceae are clearly hermaphro- ner and Ricklefs 1995), which can convolute homology ditic (plesiomorphic as scored here), apparently de- assessment. A change to drier habitats can also result 666 SYSTEMATIC BOTANY [Volume 29 in subsequent shifts to wind pollination due to loss of Herbarium (MO) and Gray Herbaria (GH) for access to herbarium pollinators (Sakai et al. 1995). Initial loss of insect pol- leaf tissue and collections. We also wish to thank J. Rodman, K. Karol, and C. Quinn for sharing DNAs, M. Fishbein for providing lination may lead to selfing and inbreeding depression, leaf material, L. Kers for first suggesting Forchhammeria might be which in turn may favor the spread of females (Char- placed outside of Capparaceae based on morphological evidence, lesworth and Charlesworth 1978). Four of the five di- K. Elliot for graphical support, S. Smith and M. S. Heschel for oecious lineages sampled occur in dry or saline habi- constructive comments, the systematics section of the Botany De- partment for discussions, and J. Rodman and an anonymous re- tats suggesting a correlation with water stressed hab- viewer for their helpful comments. We gratefully acknowledge the itats (Fig. 4; see also Sakai et al. 1995). Nonetheless, financial supports of NSF grant DEB-0073204, Garden Club of presence in dry habitats may occur without changes in America Scholarship in Tropical Botany, American Society of Plant reproductive systems since the hermaphroditic Rese- Taxonomists graduate student award, Society of Systematic Biol- daceae are distributed in arid regions. Alternatively, if ogists graduate student award, and the Davis Fund from the Bot- any Department, University of Wisconsin. the ancestral condition of the GRFT clade was dioe- cious, then reversal to hermaphroditism occurs with- out changes to more mesic habitats. Perianth reduction LITERATURE CITED is often associated with wind pollination, which is cen- AIRY SHAW, H. K. 1965. Diagnoses of new families, new names, tral to this hypothesis on the evolution of dioecy (Sakai etc., for the seventh edition of Willis’s ‘Dictionary’. Kew Bul- et al. 1995). Renner and Ricklefs (1995) argued that letin 18: 249–273. abiotic pollination favors unisexual flowers (less likely ALVERSON,W.S.,KAROL,K.G.,D.A.BAUM,M.W.CHASE,S.M. to self pollinate), which facilitates the transition from SWENSEN,R.MCCOURT, and K. J. SYTSMA. 1998. Circumscrip- tion of the Malvales and relationships to other Rosidae: evi- monoecy to dioecy. Four of the dioecious groups (Batis, dence from rbcL sequence data. American Journal of Botany 85: Apophyllum, Forchhammeria, and Gyrostemonaceae) lack 876–887. petals. Developmental studies support independent or- ———, R. NYFFELER,B.WHITLOCK,C.BAYER, and D. A. BAUM. igins of sexual systems, which correspond to correla- 1999. Phylogenetic analysis of the core Malvales based on tions of drier habitat and wind pollination. of sequences of ndhF. American Journal of Botany 86: 1474–1486. ANDERSSON,L.andS.ANDERSSON. 2000. A molecular phylogeny Pentadiplandra and Forchhammeria are unisexual as the of Tropaeolaceae and its systematic implications. Taxon 49: result of organ abortion (Hansen 1977; Ronse Decraene 721–736. 2002), whereas there is no indication of staminal ini- APG (ANGIOSPERM PHYLOGENY GROUP). 1998. An ordinal classi- tiation in floral development studies of female flowers fication for the families of flowering plants. Annals of the Mis- souri Botanical Garden 85: 531–553. of Gyrostemon and (Gyrostemonaceae; Huf- ———. 2003. An update of the Angiosperm Phylogeny Group ford, 1996). classification for the orders and families of flowering plants: Taxonomic Implications. As suggested by Kers APG II. Botanical Journal of the Linnean Society 141: 399–436. (2003), Tirania and Forchhammeria should no longer be APPEL, O. and I. A. AL-SHEHBAZ. 2003. Cruciferae. Pp. 75–174 in classified as Capparaceae s. lat. Although we have The families and genera of vascular plants: V. Flowering plants, : Malvales, Capparales, and non-betalain Caryophylla- clearly demonstrated that these taxa belong to the les, eds. K. Kubitzki and C. Bayer. Berlin: Springer. GRFT clade, not the Brassicaceae s. lat. lineage (Cap- ——— and C. BAYER. 2003. Tovariaceae. Pp 397–399 in The families paraceae, Cleomaceae, Brassicaceae), we hesitate to and genera of flowering plants: V. Flowering plants, Dicotyledons: make any formal taxonomic changes at this point in Malvales, Capparales, and non-betalain Caryophyllales, eds. K. Ku- bitzki and C. Bayer. Berlin: Springer. time. First, further sampling of members of tribe Stix- APPELQUIST,W.L.andR.S.WALLACE. 2001. Phylogeny of the eae that are likely to be part of the GRFT clade based portulaceous cohort based on ndhF sequence data. Systematic on morphological features are needed. Second, the ex- Botany 26: 409–416. act placement of Tirania relative to Forchhammeria has BARKER,F.K.andF.M.LUTZONI. 2002. The utility of the incon- not been established. As argued earlier (Chandler and gruence length difference test. Systematic Biology 51: 625–637. BARRETT, S. C. H. 2002. The evolution of plant sexual diversity. Bayer 2000), Emblingia is distinct and should be rec- Nature Reviews Genetics 3: 274–284. ognized as a monotypic family. Likewise, Pentadiplan- BAUM,D.A.,K.J.SYTSMA, and P. C. HOCH. 1994. A phylogenetic dra and Tovaria are each morphologically distinct and analysis of Epilobium (Onagraceae) based on nuclear ribosom- isolated within the core Brassicales and merit familial al DNA sequences. Systematic Botany 26: 406–419. BAYER,C.andO.APPEL. 2003a. Pentadiplandraceae. Pp. 329–331 status. With additional recognition of a family for in The families and genera of vascular plants: V. Flowering plants, Forchhammeria (with or without other Stixeae), the core dicotyledons: Malvales, Capparales, and non-betalain Caryophylla- Brassicales would be composed of a high number of les, eds. K. Kubitzki and C. Bayer. Berlin: Springer. small families. Despite discouragement of such prac- ——— and ———. 2003b. Akaniaceae. Pp. 21–24 in The families tice (APG 1998), the core Brassicales is a clade that and genera of vascular plants: V. Flowering plants, dicotyledons: Malvales, Capparales, and non-betalain Caryophyllales, eds. K. Ku- merits a number of small families as the lineages are bitzki and C. Bayer. Berlin: Springer. phylogenetically isolated, morphologically distinct, ——— and ———. 2003c. Bataceae. Pp. 30–32 in The families and and geographically disjunct (see Iltis 1999). genera of vascular plants: V. Flowering plants, dicotyledons: Mal- vales, Capparales, and non-betalain Caryophyllales, eds. K. Ku- ACKNOWLEDGEMENTS. The authors wish to thank the curators bitzki and C. Bayer. Berlin: Springer. of Wisconsin State Herbarium (WIS), Missouri Botanical Garden BUCKLEY,T.R.,P.ARENSBURGER,C.SIMON, and G. K. CHAMBERS. 2004] HALL ET AL.: PHYLOGENETICS OF CORE BRASSICALES 667

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Accession list of taxa and GenBank numbers used in phyloge- PALMER. 1993. Nucleotide sequences of the rbcL gene indicate netic analyses. The same vouchers and DNAs were used in this monophyly of mustard oil plants. Annals of Missouri Botanical study, Hall et al. 2002, and Rodman et al. 1993, 1994. Garden 80: 686–699. ———, P. S. SOLTIS,D.E.SOLTIS,K.J.SYTSMA, and K. G. KAROL. BRASSICALES 1998. Parallel evolution of glucosinolate biosynthesis inferred Bataceae: Batis maritima L. (Iltis 30500, WIS; rbcL [L22438, Rod- from congruent nuclear and plastid gene phylogenies. Amer- man et al. 1994]; ndhF [AY122403, Hall et al. 2002]; matK AY483219) ican Journal of Botany 85: 997–1006. Bretschneideraceae: Bretschneidera sinensis Hemsl. (Leu & Lin RONSE DE CRAENE, L. P. 2002. Floral development and anatomy 726, WIS; rbcL [M95753, Rodman et al. 1993]; ndhF AY483247; matK of Pentadiplandra (Pentadiplandraceae): a key genus in the AY483220) identification of floral morphological trends in the core Brass- Caricaceae: Carica papaya L. (WIS Botanical Garden; rbcL icales. Canadian Journal of Botany 80: 443–459. [M95671, Rodman et al. 1993]; ndhF AY483248; matK AY483221) ———, J. DE LAET, and E. F. SMETS. 1998. Floral development and Koeberliniaceae: Zucc. (Al Shehbaz s.n., MO; anatomy of Moringa oleifera (Moringaceae): what is the evi- rbcL [L14600, Rodman et al. 1993]; ndhF AY483249; matK dence for a Capparalean or Sapindalean affinity? Annals of AY483222) Botany 82: 273–284. Moringaceae: Moringa oleifera Lam. (Iltis 30501, WIS; rbcL ———, T. Y. A. YANG,P.SCHOLS, and E. F. SMETS. 2002. Floral [L11359, Rodman et al. 1993]; ndhF [AY122405, Hall et al. 2002]; anatomy and systematics of Bretschneidera (Bretschneidera- matK AY483223) ceae). Botanical Journal of the Linnean Society 139: 29–45. Salvadoraceae: Salvadora angustifolia Turrill (rbcL [U38532, Rod- SAKAI,A.K.,W.L.WAGNER,D.M.FERGUSON, and D. R. HERBST. man et al. 1996]) 2004] HALL ET AL.: PHYLOGENETICS OF CORE BRASSICALES 669

Tropaeolaceae: Tropaeolum majus L. (rbcL [L14706, Price and AY483234); refracta Engelm. (Vanderpool 1340, OKL; rbcL Palmer 1993]; Rodman 529, WIS; ndhF [AY122408, Hall et al. 2002]; AY483271; ndhF [AY122391, Hall et al. 2002]; matK AY483235) matK AY483224) Gyrostemonaceae: Gyrostemon sp. (Cranfield, PERTH no. 02068672; rbcL [L22439 Rodman et al. 1994]; ndhF AY483252; matK AY483236); Gyrostemon tepperi (F. Muell. ex. H. Walter) A. S. George CORE BRASSICALES (Thompson, 2243, MO; rbcL [L22440, Rodman et al. 1994]; ndhF AY483253; matK AY483237); Tersonia cyathiflora (Fenzl) A.S.George Brassicaceae s. str.: Aethionema grandiflora L. (ndhF [AF064657, (Cranfield PERTH no.02068682; rbcL [L22441, Rodman et al. 1994]; Galloway et al. 1998]; matK [AF144354, Koch et al. 2001]); Aethi- ndhF [AF122404, Hall et al. 2002]; matK AY483238) onema saxatile R. Br. (Moore s.n., WIS; rbcL AY483262; ndhF Pentadiplandraceae: Pentadiplandra brazzeana Baill. (rbcL AY483250); Arabidopsis thaliana (L.) Heynh. (rbcL [U91966, Zhu et [U38533, Rodman et al. 1996]; Hall 263, WIS; ndhF AY483254; matK al. 1997]; ndhF [AY122394, Hall et al. 2002]; matK [AF144348, Koch AY483239) et al. 2001]); Capsella bursa-pastoris (L.) Medicus (rbcL [D88904, Tsu- Resedaceae: MacBride; (Ertter 5613, WIS; rbcL kaya et al. 1997]; ndhF [AY122396, Hall et al. 2002]; matK, C. rubella AY483272; ndhF AY483255; matK AY483240); L. (Rod- Reuter [AF144334, Koch et al. 2001]); Nasturtium officinale R.Br. man 535, WIS; rbcL AY483273; ndhF [AY122406, Hall et al. 2002]; (rbcL [AF020325, Les et al. 1994]; Stahmann 233, WIS; ndhF matK AY483241) [AY122399, Hall et al. 2002]; matK AY483225); Stanleya pinnata Tovariaceae: Tovaria pendula Ruiz. & Pav. (Smith and Smith 1834, (Pursh) Britton (Hall 1, AZ; rbcL AY483263; ndhF [AY122401, Hall WIS; rbcL [M95758, Rodman et al. 1993]; ndhF [AY122407, Hall et et al. 2002]; matK AY483226) al. 2002]; matK AY483242) Capparaceae s. str.: Apophyllum anomalum F. Muell. (Covery ANOMALOUS GENERA 12044, MO; rbcL AY483264; ndhF [AY122356; Hall et al. 2002]; matK AY483227); Capparis hastata Jacq. (Iltis 30330, WIS; rbcL [M95754, Emblingia calceoliflora F. Muell. (rbcL [AF146014, Chandler and Bay- Rodman et al. 1993]; ndhF [AY122366, Hall et al. 2002]; matK er 2000]; Quinn 288a; ndhF AY483256) AY483228); Crateva palmeri Rose (Hall 105, WIS; rbcL AY483265; Forchhammeria pallida Liebmann (Iltis 29350a, WIS; rbcL AY483274; ndhF [AY122381, Hall et al. 2002]) ndhF [AY122370, Hall et al. 2002]; matK AY483229); Maerua kirkii Forchhammeria sessilifolia Standl. (Cochrane 12967, WIS; rbcL (Oliv.) F. White (Hall 261, WIS; rbcL AY483266; ndhF [AY122378, AY483275; ndhF AY483257; matK AY483243) Hall et al. 2002]; matK AY483230) Forchhammeria sp. nov. (Iltis 30784, WIS; rbcL AY483276; ndhF Cleomaceae: Cleome pilosa Benth. (Iltis 30585, WIS; rbcL AY483258; matK AY483244) AY483267; ndhF [AY122385, Hall et al. 2002]; matK AY483231); Cle- Forchhammeria trifoliata Radlkofer (Hansen 3002, WIS; rbcL ome viridiflora Schreb. (Solomon s.n., MO; rbcL AY483268; ndhF AY483277; ndhF AY483259; matK AY483245) [AY122386, Hall et al. 2002]; matK AY483232); Podandrogyne chiri- Forchhammeria watsonii Rose (Fishbein 3070, WS; rbcL AY483278; quensis (Standl.) Woodson (Nepokroeff 450, WIS; rbcL AY483269; ndhF AY483260; matK AY483246) ndhF [AY122393, Hall et al. 2002]; matK AY483233); Polanisia dode- Tirania purpurea Pierre (Squires 874, GH; rbcL AY483279; ndhF candra DC (Grette 8603, WIS; rbcL AY483270; ndhF AY483251; matK AY483261)