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Systematics of Capparaceae and Cleomaceae: an Evaluation of the Generic Delimitations of Capparis and Cleome Using Plastid DNA Sequence Data1

Systematics of Capparaceae and Cleomaceae: an Evaluation of the Generic Delimitations of Capparis and Cleome Using Plastid DNA Sequence Data1

682 Systematics of and : an evaluation of the generic delimitations of and using plastid DNA sequence data1

Jocelyn C. Hall

Abstract: The phylogenetic relationships in Capparaceae and Cleomaceae were examined using two plastid , ndhF and matK, to address outstanding systematic questions in the two families. Specifically, the monophyly of the two type genera, Capparis and Cleome, has recently been questioned. Capparaceae and Cleomaceae were broadly sampled to assess the generic circumscriptions of both genera, which house the majority of for each . Phylogenetic reconstruc- tions using maximum parsimony and maximum likelihood methods strongly contradict monophyly for both type genera. Within Capparaceae, Capparis is diphyletic: the sampled species belong to two of the five major lineages recovered in the family, which corresponds with their geographic distribution. One lineage contains all sampled New World Capparis and four other genera (Atamisquea, Belencita, , and ) that are distributed exclusively in the New World. The other lineage contains Capparis species from the Old World and Australasia, as well as the Australian , Apo- phyllum. Species of Cleome are scattered across each of four major lineages identified within Cleomaceae: (i) Cleome in part, Dactylaena, Dipterygium, Gynandropsis, , and ;(ii) Cleome droserifolia (Forssk.) Del.; (iii) Cleome arabica L., and Cleome ornithopodioides L.; and (iv) Cleome in part, , Isomeris, , and . Resolution within and among these major clades of Cleomaceae is limited, and there is no clear correspond- ence of clades with geographic distribution. Within each family, morphological support and taxonomic implications of the molecular-based clades are discussed. Key words: Capparaceae, Cleomaceae, , cpDNA, chloroplast, phylogeny. Re´sume´ : Afin d’e´tudier les questions de syste´matique en suspens dans les familles des Capparaceae et des Cleomaceae, l’auteur a examine´ leurs relations phyloge´ne´tiques en utilisant deux ge`nes plastidiques, ndhF et matK.Re´cemment, on a spe´cifiquement remis en question la monophylie des deux genres types, Capparis et Cleome. L’auteur a largement e´chan- tillonne´ les Capparaceae et les Cleomaceae, afin de de´finir les circonscriptions de ces deux genres he´bergeant la majorite´ des espe`ces de chaque famille. Les reconstructions phyloge´ne´tiques a` l’aide des me´thodes de parcimonie et de probabilite´ maximale rejettent la monophylie pour les deux genres types. Chez les Capparaceae, Capparis est diphyle´tique; les espe`ces e´chantillonne´es appartiennent a` deux des cinq ligne´es principales de´couvertes pour la famille, lesquelles correspondent avec leur distribution ge´ographique. Une ligne´e contient tous les e´chantillons de Capparis du Nouveau Monde et quatre autres genres (Atamisquea, Belencita, Morisonia et Steriphoma) distribue´s exclusivement dans le Nouveau Monde. Les au- tres ligne´es contiennent des espe`ces de Capparis de l’Ancien monde et de l’Australasie ainsi que le genre australien Apo- phyllum. Les espe`ces de Cleome se re´partissent parmi les quatre ligne´es identifie´es au sein des Cleomaceae: (i) Cleome,en partie, Dactylaena, Dipterygium, Gynandropis, Podandrogyne et Polanisia;(ii) Cleome droserifolia;(iii) C. arabica et Cleome ornithopodioides L.; et (iv) Cleome, en partie, Cleomella, Isomeris, Oxystyli et Wislizenia.Lare´solution au sein et entre ces clades majeurs demeure limite´e et il n’y a pas de correspondance e´vidente des clades avec la distribution ge´ogra- phique. Pour chaque famille, l’auteur discute le support morphologique et les implications taxonomiques des clades base´s sur l’analyse mole´culaire. Mots-cle´s:Capparaceae, Cleomaceae, Brasicaceae, cpADN, chloroplaste, phyloge´nie. [Traduit par la Re´daction]

Introduction lies Brassicaceae, Capparaceae, and Cleomaceae have high- lighted the lack of congruence between phylogeny and the As shown for many groups across the angiosperms, recent morphological characters that have been traditionally used molecular phylogenetic analyses of the closely related fami- in subfamilial, tribal, subtribal, and generic delimitations (Hall et al. 2002; Koch et al. 2003; Sa`nchez-Acebo 2005; Received 09 September 2007. Published on the NRC Research Al-Shehbaz et al. 2006; Beilstein et al. 2006). In addition, Press Web site at .nrc.ca on 25 June 2008. all three families have a high number of monotypic or di- J.C. Hall. Department of Biological Sciences, CW 405 typic genera, which are not especially helpful for reflecting Biological Sciences Building, University of Alberta, Edmonton, evolutionary relationships (Table 1; Al-Shehbaz et al. 2006). AB T6G 2E9, Canada (e-mail: [email protected]). Often, morphologically distinct small genera are embedded 1This paper is one of a selection of papers published in the within larger genera, leaving the larger genera nonmonophy- Special Issue on Systematics Research. letic. This pattern suggests that most, if not all, of these

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Table 1. Species number (species sampled in current study) and geographic distribution of genera in Capparaceae and Cleomaceae.

No. species Taxon (no. in current study) Distribution Capparaceaea s. str. F. Muell. 1 (1) Northeastern Australia Atamisquea Miers ex Hook. & Arn. 1 (1) South America, Mexico, and southwestern Bachmannia Pax 2 (0) Southern Belencita H. Karst. 1 (1) Colombia Borthwickia W.W. Sm. 1 (0) Burma, China (Yunnan) Lam. 37 (3) Tropical and , Arabia Engl. 2 (2) Western Africa Forssk. 30 (2) Africa, Madagascar, Arabia, India, Malaysia to Australia Capparis L. 250 (27) Pantropical A. Braun & Vatke 1 (0) South and southeastern Africa L. 6 (3) Pantropical Dhofaria A.G. Mill. 1 (0) Arabia Euadenia Oliv. 3 (1) Tropical Africa Hypselandra Pax & K. Hoffm. 1 (0) Burma Forssk. 50 (2) Tropical and southern Africa to tropical Asia Morisonia L. 4 (1) Caribbean and tropical South America R. Br. ex G. Don 30 (1) Tropical and subtropical Africa Steriphoma Spreng. 8 (2) Tropical Americas Thylachium Lour. 10 (2) Eastern Africa and Madagascar Cleomaceae Buhsia Bunge 1 (0) Iran Cleome L. 200 (19) Pan- and sub-tropical Cleomella DC. 10 (2) Southwestern Dactylaena Schrad. ex Schult. f. 6 (2) West Indies and Brazil Dipterygium Decne. 1 (1) Egypt to Pakistan Gynandropsis DC. 1 (1) Africa, but pantropical weed (= L.) Haptocarpum Ule 1 (0) Eastern Brazil Isomeris Nutt. ex Torr. & Gray 1 (1) Southern North America and Mexico Oxystylis Torr. & Frem. 1 (1) Southwestern United States Podandrogyne Ducke 10 (3) Central America to Andes Polanisia Raf. 4 (1) North America (includes Cristatella Nutt.) Puccionia Chiov. 1 (0) Somalia Wislizenia Engelm. 1 (1) Southwestern North America

aTribe Stixeae has been excluded; see Hall et al. (2004) and Kers (2003) small genera need to be sampled more intensively to gain a genera of both Capparaceae (Capparis L.) and Cleomaceae better understanding of phylogenetic relationships and ge- (Cleome L.). Presented herein is a plastid DNA (cpDNA) neric delimitations within these groups. Thus, to thoroughly phylogeny that represents the broadest taxon and the largest examine the monophyly of species-rich genera, numerous character sampling to date of Capparaceae and Cleomaceae monotypic and ditypic genera that are purported to be and that specifically addresses the generic limits of Capparis closely related must also be sampled. Of nearly equal impor- and Cleome. tance is the sampling of type species in molecular studies, Cleomaceae (13 genera, Table 1) are composed of three especially of species-rich genera, to contribute to the correct traditional subfamilies of Capparaceae s. l. (per Pax and formalization of nomenclatural changes that may result from Hoffmann 1936): Cleomoideae (9 genera: Cleome, Cleo- molecular analyses. Such a study has been undertaken for mella DC., Dactylaena Schrad. ex Schult. f., Gynandropsis the Brassicaceae, where a comprehensive family-level phy- DC., Haptocarpum Ule, Isomeris Nutt. ex Torr. & Gray, logeny included a large sampling of mono- and di-typic gen- Oxystylis Torr. & Frem., Polanisia Raf., and Wislizenia En- era (Beilstein et al. 2006), and it led to a re-classification of gelm.), Dipterygioideae (Dipterygium Decne.), Podandrogy- the family (Al-Shehbaz et al. 2006). However, sampling noideae (Podandrogyne Ducke), and unplaced genera strategies used in phylogenetic studies of Capparaceae and (Buhsia Bunge and Puccionia Chiov.). The close relation- Cleomaceae have not specifically addressed this issue. ship of Podandrogyne and Cleome has been proposed and Thus, while a better understanding of how the major clades widely accepted (Iltis 1958; Cochrane 1977). In contrast, of Capparaceae and Cleomaceae are related has emerged, Dipterygium has moved between Brassicaceae and Cappara- knowledge of generic and species relationships within these ceae s. l. (Hedge et al. 1980). Inclusion of Dipterygium in families remains poor, particularly for the species-rich type Cleomaceae is supported by molecular and chemical data

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(Hedge et al. 1980; Lu¨ning et al. 1992; Hall et al. 2002). based on a combined analysis of ndhF and trnL-trnF Even with the inclusion of other subfamilies, Cleomaceae is sequence information for samples from 28 species in 13 a morphologically cohesive group. It is readily distinguished genera. This study identified five major lineages within the from Brassicaceae by the following characteristics: com- group (Hall et al. 2002). Although relationships among the pound , bracteate , zygomorphic flow- lineages were not completely resolved, there was a strong ers, six of generally the same length, and dehiscent correlation between biogeographic distribution and phylog- fruits with repla and lacking false septums. Although species eny. The largest genus in the family, Capparis, was not sup- of Cleomaceae share the floral feature of a gynophore with ported as monophyletic. Instead, two OW representatives of members of Capparaceae, they are readily identified by their the genus appear to form a lineage separate from NW spe- herbaceous habit, zygomorphic flowers with six stamens, cies, a distinction also supported by morphology (DeWolf and typically dry fruits. 1962; Elffers et al. 1964). Revisions have been conducted Understanding generic relationships in Cleomaceae has on species surrounding the type species Capparis been problematic due, in part, to difficulties in establishing spinosa L. (Inocencio et al. 2005, 2006); these studies incor- the generic boundaries of the largest genus, Cleome (ca. porated genetic fingerprinting data. In addition, some Neo- 200 species). Specifically, recent analyses suggest that four tropical Capparis species have been separated into several genera, Dactylaena, Dipterygium, Podandrogyne, and Pola- genera (Iltis and Cornejo 2007a, 2007b). However, relation- nisia, may be embedded within Cleome. Phylogenetic stud- ships between OW and NW Capparis have not been explic- ies using sequences of ndhF, which codes for a subunit of itly addressed. the plastid NADH dehydrogenase enzyme, and trnL-trnF, The primary goal of this study is to improve phylogenetic which are tRNA genes, indicated that Polanisia is sister to resolution within Capparaceae and Cleomaceae. To address a clade composed of Cleome, Dipterygium, and Dactylaena the generic limits and species relationships of Capparis and (for trnL-trnF only; Hall et al. 2002). Monophyly was not Cleome, I have greatly increased taxon and character sam- supported for Cleome, despite limited sampling within the pling relative to previous studies (Hall et al. 2002, 2004), genus (four species). Two more recent studies have exam- which focused more on interfamilial relationships. Two plas- ined more species of Cleome (Sa`nchez-Acebo 2005; tid regions have been shown to be informative at discerning L.A. Inda, P. Torrecilla, P. Catala´n, and T. Ruiz, unpub- relationships among genera: ndhF and matK, the latter of lished data, 2007). Designating Polanisia as the outgroup, which encodes maturase K (Cameron et al. 2001; Davis et Sa`nchez-Acebo (2005) analyzed 34 species of Cleome. Mor- al. 2001; Hall et al. 2002; Sytsma et al. 2004; Beilstein et phology and variation in trnH-psbA sequences supported the al. 2006). The objectives of the phylogenetic analysis pre- inclusion of Podandrogyne within Cleome (Sa`nchez-Acebo sented here are to (i) assess the nature of nonmonophyly in 2005). Sampling 39 species of Cleome and using three spe- the two largest genera, Capparis and Cleome;(ii) evaluate cies of Brassicaceae as outgroups (L.A. Inda, P. Torrecilla, relationships between OW and NW representatives of Cap- P. Catala´n, and T. Ruiz, unpublished data, 2007)), however, paraceae and Cleomaceae; and (iii) elucidate the generic de- show that both Polanisia and Podandrogyne are nested limitations and species relationships within the two families. within Cleome, based on ITS (the internal transcribed The taxonomic implications of molecular-based hypotheses spacers of 18S-26S nuclear ribosomal DNA) and morphol- are also discussed, with emphasis on comparison with pre- ogy, suggesting that the use of Polanisia as an outgroup for vious sectional classifications and recent nomenclatural the study of Cleome may lead to an erroneous rooting of the changes. genus (cf. Sa`nchez-Acebo 2005). These studies, combined with other morphological and cytological information, have Materials and methods led to a new classification of the New World (NW) Cleoma- ceae (H.H. Iltis and T.S. Cochrane, unpublished data, 2008) Taxon sampling and nomenclatural changes (Iltis and Cochrane 2007). How- Taxon sampling included multiple representatives of the ever, examination of more Old World (OW) species is war- species-rich, potentially polyphyletic genera Capparis and ranted (Sa`nchez-Acebo 2005), since both molecular studies Cleome. Species were selected based on geography, morpho- were biased towards NW taxa, including only two to four logical variation, and availability of material. In addition, OW representatives. In addition, neither study sampled the taxon sampling was directed towards adding species for genera Dipterygium or Dactylaena, which are also closely which only one representative species was previously related to species of Cleome (Hall et al. 2002). Finally, no examined (Capparaceae: Boscia Lam., Buchholzia Engl., molecular study has included the type species, Cleome orni- Steriphoma Spreng.; Cleomaceae: Dactylaena, Cleomella). thopodioides L., the inclusion of which will help discern the Within Cleomaceae, 32 accessions representing 31 species correct application of the name Cleome. Thus, increased were sampled from 10 genera (Table 2). Within Cappara- sampling of genera and OW species will likely contribute to ceae, 49 accessions representing 48 species were sampled our knowledge of generic boundaries in Cleome. from 14 genera (Table 2). From the large genera Cleome In contrast to Brassicaceae and Cleomaceae, Capparaceae and Capparis, 18 and 26 species were sampled, respectively. (19 genera, Table 1) lack clear morphological synapomor- The type species of each genus (and family), Cleome orni- phies, despite molecular support for their monophyly (Hall thopodioides and Capparis spinosa, were included. Six rep- et al. 2002, 2004). However, a suite of plesiomorphic traits resentatives of Brassicaceae were included in all analyses, characterizes the family: woody habit, mostly actinomorphic since this family is sister to Cleomaceae (Hall et al. 2002). flowers with numerous stamens and a gynophore, and fleshy This sampling included saxatile R. Br. (Aethio- fruits. The broadest phylogenetic study of Capparaceae was nemeae), a member of the lineage known to be sister to all

# 2008 NRC Canada Hall 685 other tribes in Brassicaceae (Beilstein et al. 2006). The fol- addition replicates, holding three trees at each stepwise lowing outgroups were selected based on previous molecular addition, saving all minimal length trees (Multrees), and analyses of (Rodman et al. 1994, 1996, 1998; tree-bisection and rearrangement (TBR) branch swapping. Hall et al. 2004): trifoliata Radlkofer (core Because I did not run searches to completion for these Brassicales), brazzeana Baill. (Pentadiplan- search settings (owing to the existence of excessive num- draceae), cyathiflora (Fenzl.) A.S. George (Gyro- bers of most parsimonious trees in preliminary runs), and stemonaceae), and pendula Ruiz. & Pav. instead used an alternative strategy to improve the effec- (Tovariaceae). tiveness of the heuristic searches. The search was limited to saving only 10 trees per random addition replicate, and DNA extraction, amplification and sequencing the resulting consensus was then used as a backbone con- Total genomic DNA was extracted from fresh, frozen, straint to search for trees that were not consistent with the silica or herbarium samples using one of two extraction initial search. This strategy should confirm that there are methods: a modified CTAB method (Doyle and Doyle no shorter trees, and that the consensus therefore ad- 1987; Smith et al. 1991) or DNeasy Mini Kits equately represents the pool of most parsimonious trees (QIAGEN Inc., Mississauga, Ont.). Standard PCR and (Catala´n et al. 1997). All characters were treated as unor- cycle-sequencing methods were used (Hall et al. 2002, dered and equally weighted (Fitch 1971). Support for indi- 2004). The 3’-end of ndhF was amplified using 972F vidual branches was estimated using the bootstrap and 2110R primers (Olmstead et al. 1993; Hall et al. 2002; (Felsenstein 1985) as implemented in PAUP*. One thou- Beilstein et al. 2006). Four cycle-sequencing primers were sand bootstrap replicates were evaluated using the same used: 972F, 1703R, 1318F, and 2110R. The entire matK parameters as the initial unconstrained search but saving gene was amplified using trnK-710F and trnK-2R and 1000 trees per replicate. cycle-sequenced (ABI Big Dye version 3.1, Applied Bio- Maximum likelihood analyses were conducted on the cod- systems, Foster City, Calif.) with PCR primers and 495R, ing regions of individual and combined data sets. The opti- 495F, 1010R, and 1010F (Koch et al. 2001). These primers mal models of molecular evolution for individual and include the spacer regions around the matK gene, which is combined data sets were determined using the Akaike Infor- embedded in an intron between two trnK exons (Johnson mation Criterion (AIC) as implemented in Modeltest 3.7 and Soltis 1995). For some herbarium material and other (Posada and Crandall 1998). Maximum likelihood analyses problematic DNAs, ndhF and matK regions were amplified were conducted using GARLi version 0.94 (distributed by in two overlapping pieces. For ndhF, amplification used D. Zwickl at www.zo.utexas.edu/faculty/antisense/Garli. two primer pairs: 972F/1703R and 1318F/2110R, while for html) starting from random trees and using 10 000 genera- matK, the primer pairs were trnK-710F/1010R and 495F/ tions per search. ML bootstrap support values were deter- trnK-2R. All PCR products were cleaned using QiaQuick mined by conducting 100 replicates of the maximum PCR purification kits (QIAGEN). Sequencing reactions likelihood search. were purified using Performa DTR V3 96-well short plates A conditional combination approach was taken to analyz- (Edge BioSystems, Gaithersburg, Md.). Sequences were ing data from the ndhF and matK data sets. In addition to generated on an ABI3730 DNA analyzer (Applied Biosys- visually comparing trees, the incongruence length difference tems). Both strands were sequenced, with the exception of (ILD; (Farris et al. 1994) test was employed in PAUP* as a few sequences for which base-by-base proofreading of the partition homogeneity test to measure conflict between single-stranded sequence was conducted to detect any pos- individual data sets. One thousand replicates were run on sible misreads. parsimony informative characters using TBR branching Sequences were edited and initially aligned using Se- swapping with number of trees per replicate limited to quencher version 4.7 (Gene Codes Corporation, Ann Arbor, 1000. Although the ILD test has been criticized (e.g., Yoder Mich.) then codon aligned in MacClade (Maddison and et al. 2001; Barker and Lutzoni 2002), it may be used as a Maddison 2000) using the known thaliana (L.) good first test of incongruence between data sets (Hipp et al. Heynh. sequence (GenBank accession No. AP000423). As 2004). previously noted (Koch et al. 2001; Hall et al. 2002; Beilstein et al. 2006; Muller et al. 2006), both ndhF and Results matK have a relatively high frequency of insertions and (or) deletions (indels). Indels that were present in more than one Plastid sequence data accession were scored as binary characters. Parsimony anal- For the 91 taxa included in the study, the ndhF and matK/ yses were conducted including and excluding those base trnK data sets have an aligned length of 1067 bp and pairs (bp) that contain potentially informative indels, how- 2108 bp, respectively, resulting in a combined matrix of ever these regions were included in all maximum likelihood 3137 bp (Table 3). The last 37 base pairs of ndhF data set analyses (ML) analyses. were excluding from all analyses because those data were missing for most taxa. During alignment of noncoding re- Phylogenetic analysis gions between matK and trnK, some areas were difficult to Phylogenetic relationships were inferred using both parsi- align (totaling 94 bp) and these were excluded from all mony and maximum likelihood optimality criteria. The fol- analyses. Excluding or including noncoding regions of lowing parameters were used on both the individual and matK/trnK had no effect on either topology or relative combined data sets to search for multiple islands of parsimo- branch support (data not shown), so the noncoding regions nious trees in PAUP* 4.0b10 (Swofford 2002): 100 random were excluded from all subsequent analyses. Of the charac-

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Table 2. Accession table.

GenBank GenBank accession No. accession No. Taxon ndhFa matKa Voucherb/publication Capparaceae s. str. Apophyllum anomalum F. Muell. AY122356 AY483227 R. Covery & P. Hind 12044 (MO) Atamisquea emarginata Miers AY122357 EU371745 J.C. Solomon 10473 (WIS) Belencita nemorosa Jacq. AY122358 EU371746 H.H. Iltis et al. 30559 (WIS) Boscia angustifolia A. Rich. EU373677 EU371747 S.T. Pochron 15 (MO) Boscia longifolia Hadj Moust. EU373678 EU371748 P.B. Philipson et al. 3950 (MO) Boscia madagascariensis (DC.) Hadj Moust. AY122359 EU371749 Phillipson 3768 (WIS) Buchholzia coriacea Engl. AY122360 EU371750 M. Merella et al. 1656 (MO) Buchholzia tholloniana Hua EU373679 EU371751 M. Etuge 4497r (K) Cadaba kirkii Oliv. AY122361 EU371752 J. Lovett & C.J. Kayombi 4706 (MO) Cadaba virgata Boj. AY122362 EU371753 Lewis et al. 534 (MO) Capparis amplissima Lam. AY122363 EU371754 H.H. Iltis 31313 (WIS) Capparis angustifolia Kunth EU373680 EU371755 W.J. Hahn 6201 (WIS) Capparis callophylla Blume AY122364 EU371756 W.J. Hahn 6198 (WIS) Capparis crotonoides Kunth [=Capparicordis EU373681 EU371757 X. Cornejo 7586 (WIS) crotonoides (Kunth) Iltis and Cornejo] L. EU373682 EU371758 H.H. Iltis et al. 30321 (WIS) Capparis elaeagnoides Gilg EU373683 EU371759 DeChamps 11698 (MO) Capparis flexuosa L. AY122365 EU371760 H.H. Iltis 30319 (WIS) Capparis hastata Jacq. AY122366 AY483228 H.H. Iltis 30330 (WIS) Capparis indica Druce EU373684 EU371761 H.H. Iltis 30230 (WIS) Capparis lucida (Banks ex DC.) Benth. EU373685 EU371762 W.J. Hahn 6192 (WIS) Capparis odoratissima 1 Jacq. AY122367 EU371763 H.H. Iltis 30576 (WIS) Capparis odoratissima 2 Jacq. EU373686 EU371764 H.H. Iltis et al. 30526 (WIS) Capparis pachaca Kunth EU373687 EU371765 H.H. Iltis et al. 30523 (WIS) Capparis pittieri Standl. EU373688 EU371766 J.C. Hall 108 (WIS) Capparis retusa Griseb. EU373689 EU371767 E.M. Zardini & L. Guerrero 42400 (WIS) DC. EU373690 EU371768 H.H. Iltis 30502 (WIS) Capparis scabrida Kunth EU373691 EU371769 G.P. Lewis et al. 2451 (WIS) Capparis sessilis Banks ex DC. EU373692 EU371770 H.H. Iltis et al. 30561 (WIS) Capparis speciosa Griseb. EU373693 EU371771 M.C. Franceschini 50 (WIS) Capparis spinosa L. EU373694 EU371772 J. Rodman 533 (WIS) Capparis sp. nov. 1 EU373695 EU371773 H.H. Iltis & T. Ruiz 30571 (WIS) Capparis sp. nov. 2 EU373696 EU371774 H.H. Iltis et al. 30548 (WIS) Capparis stenosepala Urb. EU373697 EU371775 H.H. Iltis 30554 (WIS) Capparis tenuisiliqua Jacq. AY122368 EU371776 H.H. Iltis et al. 30512 (WIS) Lam. EU373698 EU371777 R.E. Gereau et al. 6113 (MO) Capparis verrucosa Jacq. AY122369 EU371778 H.H. Iltis et al. 30524 (WIS) L. EU373699 EU371779 S.M. Phillips & A. Weerasooriya 82 (MO) Crateva palmeri Rose AY122370 AY483229 J.C. Hall 105 (WIS) G. Forst. AY122371 EU371780 W.J. Hahn 6175 (WIS) Crateva tapia L. AY122372 EU371781 H.H. Iltis et al. 30513 (WIS) Euadenia eminens Hook. f. AY122373 EU371782 T. B. Hart 1575 (MO) DC. AY122377 EU371783 Madsen 6603 (WIS) Maerua kirkii (Oliv.) F. White AY122378 AY483230 J.C. Hall 261 (WIS) Morisonia americana L. AY122374 EU371784 H.H. Iltis et al. 30538 (WIS) Ritchiea capparoides (Andr.) Britten AY122375 EU371785 J.C. Hall 210 (WIS) Steriphoma paradoxum Endl. AY122376 EU371786 H.H. Iltis et al. 30536 (WIS) Steriphoma sp. nov. EU373700 EU371787 X. Cornejo 7345 (WIS) Thylachium africanum Lour. AY122379 EU371788 Hall 251 (WIS) Thylachium pouponii Aubre´v. & Pellegr. AY122380 EU371789 Phillipson et al. 3705 (WIS) Cleomaceae Cleome aculeata L. AY122382 EU371790 H.H. Iltis 30563a (WIS) Cleome arabica L. EU373701 EU371791 J.C. Hall sp. nov. (WIS) Cleome diffusa Banks ex DC. EU373702 EU371792 Follii 3782 (WIS)

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Table 2 (concluded).

GenBank GenBank accession No. accession No. Taxon ndhFa matKa Voucherb/publication Cleome domingensis Iltis AY122383 EU371793 DNA 2/17/89 [85-01-4] Cleome droserifolia (Forssk.) Del. EU373703 EU371794 A.G. Miller 6387 (WIS) Cleome foliosa Hook. f. EU373704 EU371795 L.E. Kers 1750 (WIS) Cleome lechleri Eichl. EU373705 EU371796 J.C. Solomon & M. Morales 17236 (WIS) Hook. subsp. jonesii (Macbr.) Iltis EU373706 EU371797 S. Vanderpool 1007 (OKL) Cleome monophylla L. AY122384 EU371798 R.E. Gereau & C.J. Kayombo 3951 (MO) Cleome ornithopodioides L. EU373707 EU371799 University of Wisconsin greenhouse Cleome oxyphylla Burch. EU373708 EU371800 L.E. Kers 3003 (WIS) Cleome parviflora Humboldt, Bonpland & Kunth EU373709 EU371801 R. Seidel 321 (WIS) ssp. psoralaeifolia (DC.) Iltis [=C. psoralaeifolia DC.] Cleome pilosa Benth. AY122385 AY483231 H.H. Iltis 30585 (WIS) Cleome rosea Vahl. ex DC. EU373710 EU371802 Ex Rio bot; JH greenhouse (WIS) 1 DC. EU373711 EU371803 University of Wisconsin greenhouse Cleome rutidosperma 2 DC. EU373712 EU371804 A.A. Mitchell 6380 (WIS) Jacq. EU373713 EU371805 G. Ayala 91-11 (WIS) Cleome viridiflora Schreb. AY122386 AY483232 Solomon s. n. (MO) L. EU373714 EU371806 J.D. Sauer 3492 (WIS) Cleomella longipes Torr. AY122387 EU371807 S. Vanderpool 1334 (OKL) Cleomella obtusifolia Torr. & Frem. EU373715 EU371808 S. Vanderpool 1293 (OKL) Dactylaena microphylla Eichler EU373716 EU371809 R.M. Harley 26503 B. Stannard & D.J.N. Hind (MO) Dactylaena pauciflora Griseb. EU373717 EU371810 J.C. Solomon & M. Nee 18108 (MO) Dipterygium glaucum Decne. EU373718 EU371811 M.I. Bajwa 972-75 (MO) Gynandropsis gynandra (L.) Briq. AY122388 EU371812 J.C. Hall 238 (WIS) [=Cleome gynandra L.] Isomeris arborea Nutt. ex Torr. & Gray AY122389 EU371813 M. Fishbein 4146 (WS) [=Cleome isomeris Greene] Oxystylis lutea Torr. & Frem. AY122390 EU371814 S. Vanderpool 1228 (OKL) Podandrogyne chiriquensis (Standl.) Woodson AY122393 AY483233 M. Nepokroeff 450 (WIS) Podandrogyne decipiens (Triana & Planch.) EU373719 EU371815 G. Mora 380 (WIS) Woodson Podandrogyne mathewsii (Briq.) Cochrane EU373720 EU371816 J.R.I. Wood 11536 (K) DC. AY483251 AY483234 D.F. Grether 8603 (WIS) Engelm. subsp. refracta AY122391 AY483235 S. Vanderpool 1340 (OKL) Brassicaceae R. Br. AY483250 EU371817 Moore. s. n. (WIS) (L.) Heynh. AY122394 AF144348 Hall et al. 2002; Koch et al. 2001 vulgaris R. Br. AY122395 EU371818 Moore 9 (WIS) oppositifolia Pers. AY122398 EU371819 Cochrane 6 Apr 2000 officinale R. Br. AY122399 AY483225 Stahmann 233 (WIS) pinnata (Pursh) Britton AY122401 AY483226 Hall 1 (AZ) Outgroups Forchhammeria trifoliata Radlkofer AY483259 AY483245 Hansen 3002 (WIS) Pentadiplandra brazzeana Baill. AY483254 AY483239 Hall 268 (WIS) Tersonia cyathiflora (Fenzl.) A.S. George AY122404 AY483238 Cranfield PERTH No. 02068682 Tovaria pendula Ruiz. & Pav. AY122407 AY483242 Smith and Smith 1834 (WIS)

aAll previously published GenBank numbers are from Hall et al. (2002, 2004), except AF144348 (Koch et al. 2001). New GenBank submissions from this study are indicated in bold. bVoucher: collector(s), collection no., herbarium code in parentheses (codes according to Holmgren and Holmgren 1998). ters in coding regions, 292 (ndhF) and 521 (matK) were Fig. 1). DNA regions containing indels were included in parsimony informative. Sixteen potentially parsimony- all analyses shown because including or excluding these informative indels of length three, six or nine bp were in- bp had no effect on topology or relative branch support troduced during alignment (five in the ndhF alignment, 11 (data not shown). For individual and combined data sets, in the matK alignment) and were mapped onto a randomly the most appropriate model of molecular evolution favored chosen MP tree using MacClade (shown on consensus in by AIC is the GTR + I + À model. This model allows for

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Fig. 1. Strict consensus tree of 850 most parsimonious trees from analysis of combined ndhF and matK data. MP bootstrap percentages based on 1000 replicates are given above the branches. Boxes on branches indicate indel events inferred for matK and ndhF; filled boxes represent single origins, open boxes multiple events. Within Capparaceae and Cleomaceae, the general geographical distributions of taxa are indicated after species names: AF, Africa and Madagascar; AS, Southeast Asia; AU, Australia; NWte, New World temperate; NWtr, New World tropics; OWte, northern Old World temperate; and asterisk (*), pantropical weed. Abbreviations for informal clade names are as follows: AF, African; NW, New World; West. N.A., western North American.

Table 3. Data set characteristics.

ndhF matK Combined General characteristics Raw length (bp) 770–968 1425–1839 n/a Aligned length (bp) 1067 2108a, 1614b 2643a Parsimony Variable sites (%) 454 (44%) 966 (48%), 800 (49%)b 1254 (47%)b Parsimony-informative sites (%) 292 (28%) 616 (30%), 521 (32%)b 813 (31%) No. of parsimony informative indels 5 11 16 Maximum likelihood Model chosen with AIC GTR+ I + À GTR + I + À GTR + I + À

aSome regions (ca. 94 bp) were unalignable. These sites were excluded from all analyses, but included here in the aligned lengths. bCoding regions only. independent rates of substitution for all nucleotide pairs remaining Capparaceae (100% BS). Relationships among (the general time reversible model, GTR) and among-site the other four major clades are not resolved or well sup- rate heterogeneity is modeled by allowing some sites to be ported, although there is weak support for a sister relation- invariant (the I parameter) while the rest have rates drawn ship between Buchholzia, a west African genus, and the AF from a discrete approximation to a gamma distribution (the capparoid clade (MP: 59% BS; ML: 69% BS). alpha parameter of the gamma shape distribution). Four major clades identified and informally named within Not unexpectedly, the ndhF and matK data sets have sim- Cleomaceae are: (i) western North American cleomoids, ilar phylogenetic structure as determined by visual inspec- comprising Cleome lutea Hook., Cleomella, Isomeris, Oxy- tion of trees and the ILD test (P = 0.2280), thus only stylis, and Wislizenia;(ii) Cleome droserifolia (Forssk.) combined cpDNA analyses are presented here (Figs. 1 and Del.; (iii)aCleome clade, including the type species of the 2). The maximum parsimony (MP) analyses resulted in genus name, C. ornithopodioides, and Cleome arabica L.; 850 MP trees with length of 3153 (consistency index = and (iv)aPolanisia clade, comprising all remaining species 0.581, retention index = 0.802). The strict consensus tree of Cleome and the genera Dactylaena, Dipterygium, Gynan- with bootstrap branch support is shown (Fig. 1). The maxi- dropsis, Podandrogyne, and Polanisia. Within the Polanisia mum likelihood search of cpDNA resulted in a single opti- clade, which is informally named based on the priority of mal tree (ln = –23654.65813; Fig. 2), which is very similar the name Polanisia, two additional clades have been recog- in topology to that recovered in MP analysis (Fig. 1). nized: Tarenaya and Andean (cf. Sa`nchez-Acebo 2005). Re- lationships among the four clades are unclear and differ Phylogenetic relationships between the analyses. ML analyses moderately support sev- Both parsimony and maximum likelihood trees strongly eral relationships (Fig. 2) that are not resolved in the MP support the monophyly of the families Brassicaceae consensus (Fig. 1): (i) Cleome droserifolia as sister to the (100% BS), Cleomaceae (100% BS), and Capparaceae (MP: western North American cleomoids (81% BS); (ii) Polanisia 99% BS, Fig. 1; ML: 98% BS, Fig. 2); the sister relationship as sister to all remaining members of the Polanisia clade of Brassicaceae and Cleomaceae is also strongly supported (76% BS); (iii) Cleome viscosa L. as sister to all other mem- (100% BS). Within Capparaceae, five major clades (all with bers of the Polanisia clade except Polanisia (87% BS), and 100% BS) are identified and are given informal names here (iv) Dactylaena as sister to Tarenaya and Andean (82% BS). to simplify discussion: (i)aCrateva clade, comprising Cra- teva L. and Euadenia Oliv.; (ii) Buchholzia;(iii) African Discussion (AF) capparoids, comprising Boscia, Cadaba Forssk., Maerua Forssk., Ritchiea R.Br., and Thylachium Lour.; Increased taxon sampling has led to a better understand- (iv) NW capparoids, including the NW species of Capparis ing of relationships within Capparaceae and Cleomaceae. and other NW genera, Atamisquea Miers, Belencita The current study has confirmed the diphyly of Capparis H. Karst., Morisonia L., and Steriphoma; and (v)aCapparis and uncovered the polyphyly of Cleome. The lack of mono- clade, including the type species of the genus name, other phyly of either genus is not necessarily surprising, especially OW species of Capparis, and the monotypic Australian ge- considering the taxonomic history of the two genera. Like nus Apophyllum F. Muell. The Crateva clade is sister to all many species-rich genera, Capparis and Cleome have been

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Fig. 2. The shortest maximum likelihood tree for the analysis of combined ndhF and matK data (ln = –23654.65813) under the GTR + I + G model. Branches are proportional to the number of changes. ML bootstrap percentages based on 100 replicates are given above the branches. Taxa with stellate and (or) lepidote are indicated by an asterisk (*); the star indicates a well-supported (99% BS) clade of taxa with this type. Sectional affiliations of Capparis (Pax and Hoffmann 1936) are provided after taxon names: BRE, Brey- niastrum; BUS, Busbeckea; CAL, Calanthea; CAP, Capparis (=Eucapparis); CAD, ; COL, Colicodendron; CYN, Cyno- phalla; MON, Monostichocalyx; QUA, Quadrella. Abbreviations for informal clade names are as follows: AF, African; NW, New World; West. N.A., western North American. Names in the Polanisia clade follow Sa`nchez-Acebo (2005). divided into sections (De Candolle 1824; Pax and Hoffmann the two groups differ in habit, fruit, and indumentum type 1936; Iltis 1952). De Candolle (1824) divided species of (DeWolf 1962). Furthermore, NW and OW species are con- Capparis into 5 sections (1 OW, 4 NW). The number of sistently placed in separate sections of the genus, regardless sections was expanded to 14 by Pax and Hoffmann (1936), of classification (De Candolle 1824; Bentham and Hooker with recent changes to species circumscription of OW sec- 1862; Pax and Hoffmann 1936). Australasian and OW spe- tions (Jacobs 1965; Inocencio et al. 2006). It should be cies of Capparis are closely related to the type species, Cap- noted that Hutchinson (1967) elevated some NW species of paris spinosa, and a genus corresponding to this clade would Capparis to the generic level, based on aestivation of the ca- retain the name Capparis. New World species of Capparis lyx (restricting Capparis to species with imbricate buds), but need new generic name(s) that will entail revisiting the clas- this classification system was not widely accepted and has sification suggested by Hutchinson (1967; see below). been shown to be artificial at the tribal level (Hall et al. 2002). Pax and Hoffmann (1936) also divided species of Old World/Australasian Capparis Cleome into sections. However, unlike Capparis, these divi- sions are not widely used in the literature, with the notable Relationships among Capparis species are well resolved; exception of that applied to NW species. Iltis (1952) divided however, current sampling in this species-rich group is lim- NW species of Cleome into seven sections, which provided ited. The monotypic genus Apophyllum, endemic to Aus- a valuable framework for recent molecular studies (Sa`nchez- tralia, is strongly supported as sister to Capparis callophylla Acebo 2005; L.A. Inda, P. Torrecilla, P. Catala´n, and Blume and Capparis tomentosa Lam., distributed in Malay- T. Ruiz, unpublished data, 2007). Most recently, NW sia and Africa, respectively. Apophyllum has a distinct mor- Cleome have been divided into a number of genera (Iltis phology: of Apophyllum anomalum F. Muell. are and Cochrane 2007). When appropriate, based on current almost leafless and dioecious. Within Capparaceae, stipular taxon sampling, previous taxonomic hypotheses of Capparis spines are usually only found in species of OW Capparis. and Cleome are compared to the cpDNA topology in the However, they also may be present in Apophyllum (Hewson discussion below. 1982), making stipular spines a candidate as a putative synapomorphy for the clade. It is interesting to note that Phylogenetic relationships within Capparaceae Dhofaria, unsampled in this study and proposed to be a Apophyllum The additional sequence information from the cpDNA close relative of , based on the shared feature of provided here continues to support monophyly for Cappara- dioecy (Miller 1988), lacks stipular spines. ceae (MP: 99% BS; ML: 98% BS) and the recognition of Initial comparisons between the plastid DNA phylogeny five clades correlated with biogeographical distribution presented here and sectional classifications of Capparis (Hall et al. 2002). Even though morphological synapomor- (Pax and Hoffmann 1936; Jacobs 1965) indicate that the phies for the family remain elusive, the unique 6 bp insertion sections generally do not reflect phylogenetic relationships. in ndhF identified previously (Hall et al. 2002) was found in Pax and Hoffmann (1936) divided OW species into five sec- all Capparaceae accessions, and appears to be an unambigu- tions based on type, indumentum type, and se- ous molecular synapomorphy. Although the present study pal fusion and arrangement. Two sections were represented includes representatives from 70% of the genera, five genera by several species in the present study, but section Bus- are notably absent from the current study and should be in- beckia was represented here by only a single species. Nei- cluded in future analyses (Table 1: Bachmannia Pax, ther section Monostichocalyx (Capparis callophylla and Borthwickia W.W. Sm., Cladostemon A. Braun & Vatke, Capparis zeylanica L.) nor section Capparis (C. tomentosa Dhofaria A.G. Mill., and Hypselandra Pax & K. Hoffm.). and Capparis elaeagnoides Gilg) is monophyletic (Fig. 2). Jacobs (1965) reduced section Capparis to a single species, Diphyly of Capparis C. spinosa, distinguished by a large, galeate (i.e., helmet- As previously demonstrated (Hall et al. 2002), cpDNA shaped) , by moving most species to section Monosti- data strongly contradict the monophyly of traditionally cir- chocalyx. Even with these changes, section Monostichocalyx cumscribed Capparis. Old World and NW Capparis repre- is not supported as monophyletic based on the cpDNA data sent two separate lineages that are not each other’s closest presented here. Although there have been recent revisions relatives, i.e., do not form sister clades, based on the current and expansions of section Capparis (Inocencio et al. 2006), sampling of 20 species distributed in the NW tropics and other sections containing the OW species of Capparis re- seven species found in temperate OW, Africa, and Austral- main in need of more detailed phylogenetic study. asia. Even with limited OW representatives, there are other lines of evidence supporting this separation. Whether the New World capparoids OW and NW species of Capparis are congeneric has long In contrast to what is observed in other clades of Cappar- been questioned, based on morphology (Jacobs 1960; aceae, relationships within NW capparoids are largely unre- DeWolf 1962). For instance, NW species lack spines and solved, possibly because the plastid markers sequenced have

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# 2008 NRC Canada 692 Botany Vol. 86, 2008 insufficient variation at this level. However, two well- emarginata Miers has been considered a synonym of Cappa- supported clades can be compared to existing sectional ris (e.g., Franceschini and Tressens 2004) or maintained as a classifications. Pax and Hoffmann’s (1936) Capparis separate genus (e.g., Kers 2003), presumably based on floral section , which was maintained as a section by morphology, including having three appendages on the re- Hutchinson (1967), is monophyletic and represented here ceptacle. Belencita, Morisonia, and Steriphoma all are dis- by Capparis amplissima Lam., Capparis flexuosa L., Cap- tinguished from Capparis by having a fused calyx. paris hastata Jacq., Capparis retusa Griseb., Cap- Steriphoma species are unique in the family in that their paris sessilis Banks ex DC., and Capparis verrucosa Jacq. flowers possess a suite of characteristics associated with (100% BS). The following features readily characterize hummingbird pollination (except Steriphoma urbani Eg- species in this section: axillary myrmecophilous (i.e., vis- gers): zygomorphy, fused calyx forming a nectary, bright ited by ants) extrafloral nectaries or glands, corymbiform red , orange , and exserted stamens. Ovaries of , sepals in two series, and torulose fruits that are Morisonia have four placentas, whereas most other Cappara- dehiscent (Iltis 1978). The second well-supported clade ceae have two. Despite the fact that all four genera are flor- (MP: 96% BS; ML: 99% BS, starred branch in Fig. 2) ally specialized, their generic status remains uncertain based does not correspond to traditional classifications because it on current phylogeny. includes intermingled representatives from four sections of Pax and Hoffmann (1936; Fig. 2) and four genera of African capparoids Hutchinson (1967; Neocalyptrocalyx Hutch., Quadrella Old World capparoids form a separate lineage from both J.S. Presl, Colicodendron Mart., and Capparidastrum NW taxa and the Capparis clade based on current study Hutch.). However, all the members sampled have stellate and morphology (Kers 2003). Four of the five genera and (or) lepidote trichomes, a unifying feature of some sampled were represented by multiple species. Of these, NW species first identified by H.H. Iltis (University of only Boscia and Cadaba are supported as monophyletic and Wisconsin – Madison, personal communication, 2007). It have potential morphological synapomorphies. Flowers of is worth noting that stellate and (or) lepidote trichomes Cadaba have prominent adaxial glands making them distinct are also found in Atamisquea, Belencita, and Capparis pit- within the family. Boscia is a genus of tropical trees with tieri Standl., of which the latter species is placed in a flowers that lack petals, but with basally connate sepals broader stellate–lepidote clade in the ML analysis (Fig. 2), forming a rim. Similar flowers are present in Thylachium albeit with no support. All members of this broader clade (petals lacking, synsepalous), but in contrast to the flowers have uniseriate sepals, but otherwise species vary in fruit of Boscia, the calyx ruptures transversely during anthesis, shape, fruit dehiscence, and number. Seed and with the ‘‘lid’’ often remaining attached. Despite the putative other fruit characters may be another good source of synapomorphy of this unusual calyx opening, molecular data potential morphological synapomorphies within NW cap- are unclear concerning the monophyly of Thylachium. Inter- paroids. For example, two members of section Cynophalla estingly, plastid data support a close relationship of Thy- (Capparis flexuosa and C. retusa) share a suite of fruit fea- lachium to Maerua (of which some species may also lack tures (including achlorophyllous embryos) that differ signif- petals), genera considered to be distantly related to each icantly from the unplaced Capparis speciosa Griseb. other by DeWolf (1962). (Franceschini and Tressens 2004). Buchholzia, a genus from western Africa, has been de- With the exception of Cynophalla, neither elevating NW scribed as an isolated genus with unclear familial affinities sections to generic status or adopting the classification of (Kers 1986, 1987). However, molecular data provide weak Hutchinson (1967) would not result in monophyletic genera. support for a sister relationship between Buchholzia and However, recent studies suggest at least some of the sections other African genera (Figs. 1 and 2), which would place all could be monophyletic, with additional taxonomic revisions. genera from this continent and Madagascar in a single clade For example, elevation of Capparis sect. Quadrella to (with the exception of some species of the pantropical Cra- Quadrella (DC.) J. Presl has been recently recognized (Iltis teva). This result has implications for corolla evolution and Cornejo 2007b; see also Hutchinson 1967), represented within the family. If a sister relationship of Buchholzia and here by Capparis odoratissima Jacq. It has been proposed other African genera is upheld, all species in the family that that Quadrella should include Capparis angustifolia lack petals belong to this African clade. However, some A. Rich. (Iltis and Cornejo 2007a) and Capparis cyno- members of this clade have petals (e.g., Ritchiea, Cadaba, phallophora L. (H.H. Iltis, personal communication,), both in part, and Maerua, in part). The propensity for loss traditionally placed in section Colicodendron. All four of only in this clade warrants further study with increased sam- these species have linear–cylindrical fruits, characteristic of pling and would be much complemented by studies of floral Quadrella, and their monophyly is supported by plastid data development data. (MP: BS 95%; ML: BS 94%). The cpDNA evidence presented here supports the proposi- Phylogenetic relationships within Cleomaceae tion of a close relationship between NW Capparis and other The phylogeny of Cleomaceae inferred in this study is NW genera, as previously suggested based on evidence from based on the broadest sampling to date of genera and OW cpDNA (Hall et al. 2002) and morphology (Kers 2003). taxa for the family, even though other studies have exam- Four genera, Atamisquea, Belencita, Morisonia, and Steri- ined more species of Cleome (Sa`nchez-Acebo 2005; phoma, are dispersed among NW species of Capparis, L.A. Inda, P. Torrecilla, P. Catala´n, and T. Ruiz, unpub- although they vary in the resolution of their phylogenetic lished data, 2007). Three genera, Buhsia, Haptocarpum, and position and their morphological distinctiveness. Atamisquea Puccionia, are not included in this sampling (Table 1). Un-

# 2008 NRC Canada Hall 693 like patterns observed in Capparaceae, the inclusion of more tatella Nutt. (=Polanisia, see Iltis 1958). Highlighting differ- taxa suggests that there may be additional major lineages ent morphological characteristics, Iltis (1958) redefined that were not previously recognized (Hall et al. 2002). Polanisia to include species with both a large adaxial gland Whereas earlier analyses of ndhF and trnL-F indicated two and notched petals, since this combination of features is not major lineages in the clade (equivalent to the Polanisia found in Cleome. The monophyly of Polanisia is not exam- clade and Western North American cleomoids), here I iden- ined in the current study, since only one species was tify up to four noteworthy lineages based on current sam- sampled, although this representative is the type species for pling. Cleome arabica plus C. ornithopodioides and the genus name. Gynandropsis gynandra has been segre- C. droserifolia are unplaced lineages in the MP tree gated from Cleome based primarily on the presence of a (Fig. 1), although the ML topology (Fig. 2) suggests the in- long androgynophore. However, this structure is variable clusion of each into one of two clades. Relationships are across the family and long androgynophores are also found congruent with other studies where there is overlap in spe- in Cleome speciosa Raf. (unsampled in present study) and cies of the Polanisia clade (Sa`nchez-Acebo 2005; Podandrogyne (Iltis 1960). Because the validity of this L.A. Inda, P. Torrecilla, P. Catala´n, and T. Ruiz, unpub- single feature has been questioned, many authors have lished data, 2007), but with increased bootstrap support here. placed Gynandropsis gynandra in Cleome (=C. gynandra; Iltis 1960; Sa`nchez-Acebo 2005; L.A. Inda, P. Torrecilla, of Cleome P. Catala´n, and T. Ruiz, unpublished data, 2007). Iltis As expected, based on data from previous studies (Hall et (1960) noted that the androphore (i.e., the stalk supporting al. 2002; Sa`nchez-Acebo 2005; L.A. Inda, P. Torrecilla, the stamens) of G. gynandra has a unique arrangement of P. Catala´n, and T. Ruiz, unpublished data, 2007) and recent vascular strands within the androgynophore, and thus, sug- nomenclatural changes (Iltis and Cochrane 2007), Cleome is gested the species represented an isolated lineage. Most re- not supported as monophyletic. However, including a greater cently, Gynandropsis was maintained as a separate genus number of genera and OW representatives resulted in a more (H.H. Iltis and T.S. Cochrane, unpublished data, 2008), complicated (and complete) picture of generic circumscrip- which is supported here (Figs. 1 and 2). Species of Podan- tion than previously described. Along with many species of drogyne are easily recognized by having unisexual flowers Cleome, five genera, Dactylaena, Dipterygium, Gynandrop- with long androgynophores and tardily dehiscent fruits with sis, Podandrogyne, and Polanisia, formed a single clade arillate seeds (i.e., seeds with an aril appendage growing (Polanisia clade; Figs. 1 and 2). Whereas the inclusion of near the hilum). Based on molecular data and morphological Podandrogyne, Polanisia, and Gynandropsis (as Cleome gy- similarities between Podandrogyne and related species of nandra) within Cleome had been identified previously Cleome,Sa`nchez-Acebo (2005) suggested Podandrogyne (Sa`nchez-Acebo 2005; L.A. Inda, P. Torrecilla, P. Catala´n, should not be recognized as a separate genus from Cleome. and T. Ruiz, unpublished data, 2007), the phylogenetic po- In contrast, Dipterygium and Dactylaena have consistently sitions of Dactylaena and Dipterygium within this clade been maintained as separate entities from Cleome. It is inter- are novel. The close relationship of C. lutea to other mem- esting to note that species of Dactylaena differ from Cleome bers of the western North American cleomoids had been only in stamen number. Flowers of Dactylaena have one established based on allozyme data (Vanderpool et al. fertile and four sterile stamens. The species of Dipterygium 1991) and inflorescence and fruit morphology (Iltis 1957) are unusual in the family because their flowers lack gyno- and this relationship is also well supported in both MP phores and their fruits are indehiscent, a characteristic typi- and ML topologies (Figs. 1 and 2). Cleome arabica and cally only found in the North American cleomoids. the type species C. ornithopodioides either make up an Careful morphological examination of the well-supported isolated lineage (MP, Fig. 1) or represent members of the clades in Cleomaceae (Figs. 1 and 2) is necessary to identify Polanisia clade (ML, Fig. 2). The phylogenetic position of potential morphological synapomorphies for these groups, C. droserifolia within the family is also largely unclear since the characters traditionally used delimit para- or poly- (Figs. 1 and 2). In summary, species of Cleome are en- phyletic genera. Specifically, Sa`nchez-Acebo (2005) and trenched within all major lineages of Cleomaceae along H.H. Iltis and T.S. Cochrane (unpublished data, 2008) point with a number of other genera. out the potential importance of seed and characters in What then, is the morphological basis for separating gen- the family. There has been strong morphological and molec- era from Cleome? Emphasis has often been placed on two ular evidence for the close relationship of Podandrogyne to major characters, stamen number and length of the androgy- species of Cleome in the informally named group referred to nophore (the structure formed from the fusion of gynophore as Andean (Figs. 1 and 2; Iltis 1952; Sa`nchez-Acebo 2005), to staminal tube), although generic distinctions are often which is soon to be formally recognized as the genus supported by other morphological characteristics. Polanisia, Andinocleome (H.H. Iltis and T.S. Cochrane, unpublished Podandrogyne, and Gynandropsis have been subsumed ei- data, 2008), represented by Cleome lechleri Eichl. and ther within Cleome or the species compositions of these gen- Cleome pilosa Benth. in the current study. Cleome lechleri era have varied relative to Cleome, based on the perceived and Podandrogyne share a high chromosome number of n = importance of stamen number and androgynophore length. 29 (Cochrane 1978), a putative synapomorphy for the clade. De Candolle (1824) placed all Cleome species with greater In addition, Sa`nchez-Acebo (2005) stated that members of than six stamens, the typical number found in the genus, in Andinocleome and Podandrogyne are woody (except for Polanisia. In contrast, Pax and Hoffmann (1936) dismissed C. chilensis DC., unsampled in the current study) and share the importance of stamen number variation and merged all seed surface and internal characteristics. Tarenaya, another species with Cleome, except for two species placed in Cris- well-supported clade (MP:100% BS; ML 99%BS), was first

# 2008 NRC Canada 694 Botany Vol. 86, 2008 described by Iltis (1952) and its monophyly confirmed with paraceae and Cleomaceae exhibit significant floral diversity molecular studies by Sa`nchez-Acebo (2005). Most species in that is remarkable, considering how stable the floral plan is this clade have echinate pollen and similar seed morphology in over 3000 species of Brassicaceae. Cleomaceae also pro- (Sa`nchez-Acebo 2005) in addition to having spines. The vides an important comparison with Arabidopsis and other topology presented here is consistent with that of Sa`nchez- members of Brassicaceae. For example, understanding the Acebo (2005), except that her psbA-trnH data placed Gynan- evolution of both C-4 photosynthesis and genome duplica- dropsis gynandra within this clade with low bootstrap tion in Brassicales will be aided by strong phylogenetic hy- support (64% BS). Sa`nchez-Acebo (2005) highlights that potheses of Cleomaceae. Combining both phylogenetic Gynandropsis is morphologically more similar to OW spe- information and comparative genomics approaches, Schranz cies than to members of Tarenaya. The potential wealth of and Mitchell-Olds (2006) demonstrated that Cleome has taxonomically informative seed characters needs to be undergone an independent event from that within explored by studying Dactylaena and Dipterygium, genera , which provides insight into the evolution of gene newly placed in the current and previous studies (Hall et al. duplicates. Cleome is the nearest C-4 relative to Arabidopsis 2002). and shows significant promise as a model for understanding the evolution of C-4 photosynthesis (Brown et al. 2005; Taxonomic implications Voznesenskaya et al. 2007). However, clear understanding The two focal families differ significantly in the nature of of phylogenetic relationships within Cleome is necessary to nonmonophyly of type genera, resulting in different pro- make use of the C-4 system and possible transitions between posals for nomenclatural changes. All species of Capparis, C-3 and C-4 photosynthesis (Brown et al. 2005; or at least those sampled to date, are placed in one of two Voznesenskaya et al. 2007). The cpDNA phylogeny lineages in the family. All NW taxa need to be re-named, presented here demonstrates the need for such studies to in- although the potentially straightforward approach of elevat- clude many other genera in the Cleomaceae. Ultimately, to ing sections is not valid, with the possible exception of sec- understand ancestral character states of Cleomaceae and tion Cynophalla. Either all members of the NW capparoids Brassicaceae and the transitions between them, a further should be recognized under the same genus name or they comparison to Capparaceae is needed. should be divided into separate genera. There are clades with both molecular and morphological support, which sug- Acknowledgements gests Capparis species could be placed into smaller, mono- This research was funded by an Natural Sciences and En- phyletic, segregate genera (cf. Iltis and Cornejo 2007a, gineering Research Council of Canada Discovery grant and 2007b). The phylogenetic pattern emerging within Cleoma- by the Faculty of Science, University of Alberta. I would ceae is complex with regard to the circumscription of like to thank H.H. Iltis and K.J. Sytsma for collaboration on Cleome. New combinations and new genera have been pro- previous research in Capparaceae and Cleomaceae; posed for the NW species for the Flora of North America M. Beilstein and C. Davis for providing insightful comments (Iltis and Cochrane 2007). These changes contradict on an earlier version of this paper; S. Warwick and an anon- suggestions made by Sa`nchez-Acebo (2005) and L.A. Inda, ymous reviewer for their helpful feedback; P. Catala´n for P. Torrecilla, P. Catala´n, and T. Ruiz (unpublished data, sharing a submitted manuscript; H. Iltis and T. Cochrane 2007) to include Podandrogyne and Polanisia (L.A. Inda, for providing me with a copy of their unpublished manu- P. Torrecilla, P. Catala´n, and T. Ruiz, unpublished data, script and for their comments on this manuscript; 2007) within Cleome. Given the strong support for the close G. Gasline for obtaining material of Buchholzia thol- relationship of both Dipterygium and Dactylaena with spe- loniana; and the Service Unit, Depart- cies of Cleome, the approach of subsuming smaller genera ment of Biological Sciences – University of Alberta, for into Cleome would place all species of the family, possibly support with sequencing. The following herbaria generously excluding western North American cleomoids, into a single, provided loans: BM, GH, K, MO, and WIS. morphologically diverse genus. Having such morphologi- cally distinct entities in a single genus is unwieldy. How- References ever, more sampling of OW taxa is clearly needed and suggestions of nomenclatural changes based on current top- Al-Shehbaz, I.A., Beilstein, M.A., and Kellogg, E.A. 2006. Sys- ology are premature. Under current sampling, a conservative tematics and phylogeny of the Brassicaceae (Cruciferae): an approach would leave only Cleome ornithopodioides (the overview. Plant Syst. 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