American Journal of Botany 92(8): 1397±1420. 2005.

MOLECULAR PHYLOGENETIC ANALYSIS OF UNIOVULATE EUPHORBIACEAE (EUPHORBIACEAE SENSU STRICTO) USING PLASTID RBCL AND TRNL-F DNA SEQUENCES1

KENNETH J. WURDACK,2,5 PETRA HOFFMANN,3 AND MARK W. C HASE4

2Department of Biology, Coker Hall, University of North Carolina-Chapel Hill, North Carolina 27599 USA; 3Herbarium, Royal Botanic Gardens, Kew, Richmond, Surrey, TW9 3AB, UK; and 4Jodrell Laboratory, Royal Botanic Gardens, Kew, Richmond, Surrey, TW9 3DS, UK

Parsimony and Bayesian analyses of plastid rbcL and trnL-F DNA sequence data of the pantropical family Euphorbiaceae sensu stricto (s.s.) are presented. Sampling includes representatives of all three subfamilies (Acalyphoideae, Crotonoideae, and Euphorbioi- deae), 35 of 37 tribes and 179 of the 247 genera of uniovulate Euphorbiaceae sensu lato (s.l.). Euphorbiaceae s.s. were recovered as a monophyletic group with no new adjustments in circumscription. Two clades containing taxa previously placed in Acalyphoideae are found to be successive sisters to all other Euphorbiaceae s.s. and are proposed here at subfamilial rank as Peroideae and Cheilo- soideae. The remainder of the family fall into seven major lineages including Erismantheae and Acalyphoideae s.s. (parts of Acaly- phoideae), Adenoclineae s.l., Gelonieae, articulated crotonoids and inaperturate crotonoids (parts of Crotonoideae), and Euphorbioideae. Potential synapomorphies and biogeographical trends are suggested for these clades. Acalyphoideae s.s., inaperturate crotonoids, and Euphorbioideae tribe Hippomaneae each have two major subclades that represent novel groupings without apparent morphological synapomorphies. Two subfamilies, 14 tribes, and 10 genera were found to be para- or polyphyletic. Noteworthy among these, Om- phaleae are embedded in Adenoclineae, Hureae ϩ Pachystromateae in Hippomaneae, Ditta in Tetrorchidium, and Sapium s.s. in Stillingia.

Key words: Cheilosoideae; Euphorbiaceae; Malpighiales; molecular phylogenetics; morphology; Peroideae.

The order Malpighiales sensu the Angiosperm Phylogeny cluding Dicoelieae and Galearieae), Crotonoideae, and Eu- Group (APG II, 2003, but excluding Peridiscaceae following phorbioideae; Pandaceae from Acalyphoideae±Galearieae, Davis and Chase [2004] and including Raf¯esiaceae sensu Phyllanthaceae from Phyllanthoideae pro parte (excluding stricto [s.s.] [see Davis and Wurdack, 2004]) contains 29 (to Centroplaceae and Drypeteae, and including Croizatieae and 37 depending on alternative sensu stricto circumscriptions) Dicoelieae), Putranjivaceae from Phyllanthoideae±Drypeteae families of eudicots. Five of these families, Euphorbiaceae s.s., (excluding Lingelsheimia), and Picrodendraceae from Old®el- Pandaceae, Phyllanthaceae, Picrodendraceae, and Putranjiva- dioideae (excluding Croizatieae). The modern sensu stricto ceae (see Wurdack et al., 2004, for family comparisons), have family delimitation was proposed by Corner (1976) and Meeu- often been united as Euphorbiaceae sensu lato (s.l.). The cir- se (1990) to include members of Euphorbiaceae s.l. that pos- cumscription of Euphorbiaceae s.l. has long been controversial sess a single ovule per locule (instead of two), excluding Pan- (reviewed by Webster, 1987), and the current APG II splitting daceae and Scagea (secondarily uniovulate Picrodendraceae). of the group re¯ects long-known fundamental divisions that The remaining segregates (Phyllanthaceae, Picrodendraceae, were in part used for previous subfamilial circumscriptions and Putranjivaceae) possess two ovules per locule, although in (i.e., Webster, 1975, 1994b; Radcliffe-Smith, 2001). The ori- many taxa only one of the paired ovules develops into a seed. gins of the APG II segregates include, with some adjustments, Gynoecial evidence links Euphorbiaceae s.s. with Picroden- Euphorbiaceae s.s. derived from Acalyphoideae pro parte (ex- draceae and Phyllanthaceae, notably a shared and putatively unique combination of nucellar beak, epitropus ovules, and 1 Manuscript received 16 August 2004; revision accepted 6 May 2005. obturator (Sutter and Endress, 1995). Putranjivaceae appear The authors thank the following individuals who have provided plant further removed based on embryological differences (Tokuoka material: P. Berry (WIS), K. Cameron (NY), J. CedenÄo, G. Challen (K), P. and Tobe, 1999). Delprete, P. Fine, L. Gillespie (CAN), J. Horn (DUKE), J. Hughey, B. Jestrow, G. McPherson (MO), T. Motley (NY), C. Parks (NCU), S. Pell (NY), R. Single-exemplar sampling of Euphorbiaceae s.s. (Euphor- Pooma (BKF), L. Prince (RSA), V. Steinmann (IEB), and G. Webster (DAV). bia) employed in major angiosperm-wide molecular phyloge- We also thank the curators of DAV, DUKE, FTG, K, L, MEL, MO, NY, RSA, netic studies (Chase et al., 1993; Soltis et al., 1997, 2000, US, WAG, Kebun Raya, Bogor, and the Australian National Botanic Gardens 2003; Savolainen et al., 2000a; Hilu et al., 2003) has failed to (Canberra, Sydney), for permission to sample herbarium and/or living recover supported sister relationships for the family, and po- collections. Additional unpublished sequences were provided by H. Hills and lyphyly of Euphorbiaceae s.l. has been indicated. Limited ad- A. Hipp. P. Berry, W. J. Hayden, G. Levin, G. McPherson, J. Nowicke, C. Specht, V. Steinmann, G. Webster, and P. van Welzen have provided ditional sampling using rbcL (Wurdack and Chase, 1996, invaluable euphorb discussions or helpful comments on the manuscript. We 1999; Savolainen et al., 2000b; Chase et al., 2002; Wurdack, are grateful to G. Challen and J. Bates (SI) for curatorial and computer 2002) and/or other genes (Spichiger et al., 1993; Downie et support, respectively. Research by K. J. W. was generously supported by the al., 1996; Savolainen et al., 1997; Cho et al., 1998; Wurdack, Smithsonian Institution and the Lewis B. and Dorothy Cullman Program for 2002; Davis and Chase, 2004; Davis and Wurdack, 2004; Da- Molecular Systematic Studies at The New York Botanical Garden. vis et al., 2005) has revealed little about sister groups or core 5 Author for correspondence (e-mail: [email protected]) present address: Department of Botany and Laboratories of Analytical Biology, relationships in the family. Smithsonian Institution, P.O. Box 37012, NMNH MRC-166, Washington, The focus of this paper is Euphorbiaceae s.s., which com- D.C. 20013-7012 USA. prise c. 6300 species (estimated from Govaerts et al., 2000) in 1397 1398 AMERICAN JOURNAL OF BOTANY [Vol. 92

245 genera (Radcliffe-Smith, 2001) or alternatively 219 genera however, is appearing with increasing frequency, including for in the previous classi®cation adopting a broader generic con- Dalechampia (Armbruster and Baldwin, 1998; Armbruster, cept (Webster, 1994b). Two additional genera, Aubletiana and 2002), Euphorbia s.l. (Gielly and Taberlet, 1994; Savolainen Colobocarpos, have been recently recognized for aberrant spe- et al., 1997; Molero et al., 2002; Steinmann and Porter, 2002; cies of Conceveiba and Croton, respectively. Euphorbiaceae Ritz et al., 2003; Haevermans et al., 2004), Fontainea (Ros- s.s. is the largest component of Euphorbiaceae s.l. and remains setto et al., 2001), Hevea (Luo and Boutry, 1995; Luo et al., as one of the largest plant families even in this more restricted 1995), Macaranga (Blattner et al., 2001; Davies et al., 2001), circumscription. Of all Malpighiales families, Euphorbiaceae Manihot (Bertram, 1993; Olsen and Schaal, 1999), and Mer- s.s. are unsurpassed in species richness, morphological and curialis (KraÈhenbuÈhl et al., 2002) and is underway for Aca- phytochemical diversity, and economic importance. The no- lypha (Steinmann and Levin, 2003) and Croton (Berry et al., table economic products include cassava (Manihot esculenta), 2002). Mitochondrial gene-content surveys have shown poten- rubber (Hevea brasiliensis), castor (Ricinus communis) and tially useful variation among core lineages (Adams et al., tung oils (Vernicia spp.), candilla wax (Euphorbia spp.), and 2002). Genomic work has been published on Ricinus (Loo et ornamental poinsettias (Euphorbia pulcherrima). Molecular- al., 1995) and Euphorbia (Edqvist and Farbos, 2002) and is clock dating (WikstroÈm et al., 2001; Davis et al., 2005) gives underway for economically important Hevea and Manihot. a Cretaceous estimate for divergence of the family. The fossil The phylogenetic analyses of Euphorbiaceae s.s. that we record is of poor quality with few reliable Euphorbiaceae s.s. report here used DNA sequence data from the plastid regions macrofossils (see Webster, 1967; Crepet and Daghlian, 1982), rbcL, and the trnL-F spacer and trnL intron (hereafter referred although it does indicate considerable diversi®cation by the to as ``trnL-F''). The utility of rbcL for resolving intra- and Eocene (Muller, 1981; Crepet and Daghlian, 1982; Friis and interfamilial relationships is well known and has been dem- Crepet, 1987; Dilcher and Manchester, 1988). Systematics of onstrated previously in Malpighiales (i.e., Cameron et al., the family stands on the work of Webster (1975, 1994b) and 2001; Chase et al., 2002; Gustafsson et al., 2002; Wurdack, is presently inferred to be largely coincident with that of the 2002; Wurdack and Chase, 2002; Wurdack et al., 2004). The uniovulate subfamilies Acalyphoideae, Crotonoideae, and Eu- lack of bootstrap support along the spine of these trees, even phorbioideae. Webster's 1994 system also placed Pandaceae as with the addition of genes of similar or slower evolutionary tribe Galearieae of Acalyphoideae, although previously he had rates, and poor resolution among closely related taxa indicates maintained it as a family-level segregate within Euphorbiales that more rapidly evolving regions are required. Largely non- (1975; see Webster [1987] for a discussion of this change of coding trnL-F is frequently used at the species level including opinion). Dicoelia had been referred to a monotypic tribe (Di- in Euphorbiaceae s.s. (Gielly and Taberlet, 1994; Rossetto et coelieae) either in Pandaceae (Webster, 1987) or adjacent to al., 2001; Ritz et al., 2003), but its broader utility has been Galearieae (Webster, 1994b). The presence of two ovules per demonstrated (e.g., Freeman and Scogin, 1999; Richardson et locule has long indicated misplacement of Dicoelia, and a po- al., 2000; Davis et al., 2001; Bremer et al., 2002; Borsch et sition in Phyllanthaceae has recently been con®rmed (Kath- al., 2003). Alignment issues with increasing sequence diver- riarachchi et al., 2004, 2005). Relative to the classi®cation of gence can ultimately limit the higher-level usefulness of non- Webster (1994b), that of Radcliffe-Smith (2001) is nearly coding DNA (see Kelchner, 2000; Simmons and Freudenstein, identical in the composition of Acalyphoideae (20 tribes, 119 2003). Our study was undertaken to evaluate the circumscrip- genera), Crotonoideae (12 tribes, 74 genera), and Euphorbioi- tion of Euphorbiaceae s.s. and monophyly of suprageneric taxa deae (5 tribes, 54 genera). His system recognizes a number of recognized in recent classi®cations, and to elucidate patterns generic segregates that had been subsumed by Webster, con- of infrafamilial relationships and character evolution. We have tains a reevaluation and further generic splitting of Euphor- suggested modest classi®cation changes but have refrained bioideae±Hippomaneae, and changes the classi®cation of sev- from proposing a full revision of the family classi®cation be- en other genera, including the creation of a new tribe (Aca- cause of limitations due to sampling and resolution. lyphoideae±Sphyranthereae) to accommodate Sphyranthera. Systematic palynological studies have done much to guide MATERIALS AND METHODS or illuminate current classi®cations (Punt, 1962; Nowicke, 1994; Takahashi et al., 1995, 2000; FernaÂndez-GonzaÂlez and Taxon samplingÐTaxa, voucher information, and GenBank numbers for Lobreau-Callen, 1996; Lobreau-Callen and SuaÂrez-Cervera, all 213 rbcL and 221 trnL-F sequences used are listed in the Appendix. Un- 1997; Nowicke et al., 1998, 1999, 2002). The understanding published sequences include 169 newly generated rbcL and 220 trnL-F, along of evolutionary trends in Euphorbiaceae s.l. is clouded by the with 36 published rbcL sequences that were originally generated for this study belief that the uniovulate subfamilies are derived from bio- but used ®rst elsewhere (KaÈllersjoÈet al., 1998; Savolainen et al., 2000b; Chase vulate lineages (i.e., from within a paraphyletic Phyllanthoi- et al., 2002; Wurdack et al., 2004; Davis et al., 2005). Following Radcliffe- deae [ϭ Phyllanthaceae]). Similarly, many tribes and subfam- Smith (2001), sampling included 35 of 37 tribes and 179 of 247 genera at- ilies Acalyphoideae and Crotonoideae are believed to be par- tributed to the uniovulate subfamilies of Euphorbiaceae s.l. Generic names aphyletic (Webster, 1987). Evolutionary relationships among strictly follow Radcliffe-Smith (2001) for ease of reference. Phylogenetically placed here but not tallied in our generic count are the unpublished new genus uniovulate Euphorbiaceae were ®rst depicted in the intuitive Brasiliocroton (Berry et al., in press), as well as Vaupesia (data obtained phylograms of Pax (1924). A number of small morphological during revisions and not available for inclusion in ®gured analyses). Not sam- phylogenetic studies (Esser, 1994; Welzen, 1995, 1999; Kruijt, pled from lack of adequate material were one monogeneric tribe (Sphyranth- 1996; Esser et al., 1997; Welzen and Stuppy, 1999) have fo- ereae) and potentially signi®cant genera (as suggested by Webster [1994b], cused on speci®c tribes, but their sampling was constrained a Radcliffe-Smith [2001] or other evidence) Anomalocalyx, Avellanita, Chei- priori to follow the circumscriptions of Webster (1994b) and losa, Chondrostylis, and Dendrocousinsia; the remaining unsampled tribe (Di- did not address the premise of monophyly. No published mo- coelieae) has been excluded from Euphorbiaceae s.s. (see Introduction). Par- lecular phylogenetic study has focused solely on higher-level ticular effort was made to sample more thoroughly the ``basal'' tribes of Aca- relationships within Euphorbiaceae s.s. Species-level work, lyphoideae. An increased sampling of Hippomaneae was included to examine August 2005] WURDACK ET AL.ÐEUPHORBIACEAE PHYLOGENETICS 1399

TABLE 1. Data set and parsimony-based tree characteristics for rbcL and trnL-F analyses of Euphorbiaceae s.s.

Combined Characteristics rbcL trnL-F rbcL trnL-F rbcL ϩ trnL-F No. taxa 213 220 179 179 179 Matrix length 1428 2298 1428 2298 3726 Characters analyzed 1398 842 1398 842 2240 Missing data in characters analyzed (%) 1.01 5.13 0.37 4.66 1.98 Constant characters 884 281 910 292 1202 Variable characters 514 561 488 550 1038 Parsimony informative characters 379 441 355 419 774 No. trees Ͼ1 ϫ 106 Ͼ1 ϫ 106 Ͼ1 ϫ 106 Ͼ1 ϫ 106 87 862 Tree length 2358 2257 2154 2107 4288 CI (ϭ CI excluding uninformative sites) 0.306 (0.258) 0.425 (0.386) 0.315 (0.264) 0.439 (0.394) 0.374 (0.326) RI 0.706 0.785 0.676 0.749 0.711 RC (ϭ RC excluding uninformative sites) 0.216 (0.182) 0.334 (0.303) 0.213 (0.179) 0.329 (0.295) 0.266 (0.232) the generic delimitations and classi®cation proposed by Esser (Esser et al., trimmed of primer regions. Alignments of rbcL were easily managed by eye, 1997; Esser, 1999; Radcliffe-Smith, 2001, 352±398). Given the undisputed whereas trnL-F sequences were aligned with CLUSTAL W (as implemented monophyly of Euphorbia s.l. and recent well-sampled molecular phylogenetic in CLUSTAL X v1.83; Thompson et al., 1997), followed by extensive hand studies (i.e., Steinmann and Porter, 2002), sampling in the cyathial clade was re®nements in Se-Al v2.0a11 (Rambout, 1996±2002) that considered homol- intentionally low relative to its species richness. ogy assessments from the standpoint of both similarity and possible mecha- The choice of outgroup is somewhat arbitrary due to the lack of bootstrap- nism of molecular evolution (i.e., following Kelchner, 2000; Borsch et al., supported sister relationships to Euphorbiaceae s.s. in ordinal molecular phy- 2003). Indels were abundant in trnL-F. Although small groups of related taxa logenetic analyses (see Chase et al., 2002; Wurdack, 2002; Davis and Chase, were easily alignable, ambiguous regions of high complexity were present in 2004; Davis et al., 2005). Humiriaceae were chosen based upon their low the complete matrix, especially in Euphorbia s.l. Secondary structure predic- level of molecular divergence and concomitant ability to align with ingroup tion in trnL-F used mfold v3.1 on a web server (http://www.bioinfo.rpi.edu/ trnL-F sequences (see Davis et al., 2001; Wurdack, 2002). Pandaceae were applications/mfold; Zuker, 2003) with default parameters to evaluate corre- included to demonstrate their monophyly and misplacement in the classi®- spondence between structural motifs and some sequence changes (i.e., com- cations of Webster (1994b) and Radcliffe-Smith (2001), although interfamilial pensatory changes, conserved P-elements, and involvement of hairpins in in- sequence divergence in trnL-F makes homology assessments more dif®cult. versions). Mononucleotide repeats were few (six ``hotspots''), and much of Additional available Malpighiales trnL-F sequences (mostly K. J. Wurdack, the length variation involved short tandem repeats of 3±15 nucleotides. A unpublished data) include those for Achariaceae, Malpighiaceae (from Davis similarity criterion was used to associate the least divergent copies of such et al., 2001), and biovulate lineages (Phyllanthaceae, Picrodendraceae, and repeats. Often the copies were identical, in which case incorrect homology Putranjivaceae), but these proved unsatisfactory due to the introduction of assessment would have no phylogenetic impact under the data exclusion sets additional ambiguously aligned regions. used. Patterns were evident of indel-poor regions interspersed with variable regions often containing multiple, independent (non-homologous) insertions. Laboratory methodsÐDNA extraction, polymerase chain reaction (PCR) The data matrices are archived in TreeBASE (www.treebase.org/treebase/) and ampli®cation, and sequencing methods followed those previously outlined are available from the authors. (Wurdack, 2002; Wurdack et al., 2004). In some Hippomaneae, potent PCR Maximum parsimony (MP) analyses were conducted using PAUP* 4.0b10 inhibitors co-puri®ed with Qiagen DNeasy Plant Mini kit extractions (Qiagen, (Swofford, 2003) and a two-stage search strategy with starting trees generated Valencia, California, USA). Inhibition could usually be overcome by template by 1000 random taxon addition sequence replicates (RAS), equal weights and dilution. Ex Taq DNA Polymerase (hot-start version; Takara Mirus Bio, Mad- unordered characters, tree-bisection-reconnection (TBR) swapping, holding 10 ison, Wisconsin, USA) was found to be especially sensitive for degraded or trees at each step, and MulTrees (holding more than one tree) off (Wurdack weak extractions. Manual radioactive methods were used for 19% of our rbcL et al., 2004). The resulting trees were used as starting trees with MulTrees on sequences, and the rest were generated by automated ¯uorescent sequencing. and a maximum tree limit of 1 ϫ 106. We also used PAUPRat (Sikes and The trnL-F sequences were generated entirely by automated sequencing using Lewis, 2001); however, no shorter trees were found. The searches shown here four primers (c, d, e, f; Taberlet et al., 1991) after ampli®cation as one piece were done on rbcL, trnL-F, and the combined data set. The combined data (primers c ϩ f) or two pieces (c ϩ d, e ϩ f) with some degraded templates. set included six composite terminals (i.e., each partition came from a different A newly designed trnL-intron internal primer (aligned positions 272±292, trnL-intF: 5Ј-GGTGCAGAGACTCAATGGAAG-3Ј) was used as substitute DNA source but representing the same species for Croton lucidus, Cr. lobatus, for primer ``c'' with templates too degraded for ampli®cation of the entire and Maprounea guianensis, or same genus for Bernardia, Dysopsis, and Te- intron. Homopolymer regions affecting sequence quality were sporadic in both traplandra). The individual data sets included 75 (34 rbcL and 41 trnL-F) the trnL-F spacer and intron. Twenty-three sequences were ampli®ed and se- terminals that were only present in one partition and not included in the quenced as two pieces, in which case we treated the internal primer-binding combined analysis. Other analyses (not shown) included each separate parti- region (primers d, e) as missing data. Data from Bernardia scabra, Concev- tion of the combined sampling (see Table 1), combined searches with all data, eiba pleiostemona, Mareyopsis, Sampantaea, and Vaupesia could only be ob- and compartmentalized searches of large clades. Searches on subpartitions tained for the trnL-F spacer because of the degraded nature of these herbarium de®ned by the ampli®cation primers used for degraded samples showed no DNAs. Micrococca, Strophioblachia (both Chase et al., 2002), and Euphorbia bootstrap-supported incongruence that could otherwise indicate the assembly epithymoides (as E. polychroma; Chase et al., 1993) were resequenced for of chimeric sequences. The ®rst 30 bp of rbcL were excluded to reduce miss- rbcL from the original DNAs as the published sequences were found to con- ing data in the primer-binding region that was ragged due to different length tain sequencing errors or missing data. Both strands were sequenced for all primers used. Indels in trnL-F were treated as missing data, and for the ®nal taxa. analyses described here, 1468 bp of that matrix were excluded. This reduced the missing data from 56.9% to 5.13% and eliminated most ambiguously Sequence assembly and data analysisÐSequences were assembled and aligned regions, one inversion hotspot (trnL-F spacer aligned positions 1776± edited in Sequencher 3.1.1 (Gene Codes, Ann Arbor, Michigan, USA) and 1813), and most insertions. Details of the excluded regions are embedded in 1400 AMERICAN JOURNAL OF BOTANY [Vol. 92 the archived Nexus data ®les with accompanying exclusion sets. We acknowl- identi®ed, although some were not recovered intact in the rbcL edge that this conservative approach might have caused the loss of some analysis. phylogenetic information (but not necessarily; Chase et al., 2000) but believe this is better recovered (i.e., indel coding and fewer ambiguous regions to Analyses of rbcLÐThe termination codon beginning at nu- exclude) with analyses of subsets of the data from individual clades. Such cleotide 1426 was, as in most Malpighiales, predominant in compartmentalized analyses (noted earlier) were found to have little effect on Euphorbiaceae s.s., but RuBisCo length variants beyond the local topologies despite being analytically superior in terms of reduced am- typical 475 amino acids occurred (fully sequenced until stop biguous regions and more thorough search implementation. Zero-length codon or inferred from lack of one at position 1426 where branches were collapsed and uninformative characters were included in anal- some sequences were truncated) in 17 taxa and were inferred yses except, as noted, for the calculation of alternative tree statistics. Tree to be from eight separate gains. The MP strict consensus is statistics included the consistency index (CI; Kluge and Farris, 1969), reten- shown in Fig. 1. Euphorbiaceae s.s. are strongly supported, tion index (RI; Farris, 1989), and rescaled consistency index (RC; Farris, 1989). Relative clade support was evaluated with 1000 bootstrap (Felsenstein, but the spine of the tree is poorly resolved; seven of the nine 1985) replicates (split into 125 or 200 replicate blocks and tree®les subse- major lineages are recovered intact. Peroideae are not recov- quently combined using stored tree weights), each with 20 or 100 RAS using ered as a monophyletic group, and Pogonophora is sister to TBR swapping, holding 10 trees at each step, and saving no more than 10 the rest of Euphorbiaceae s.s., although this lacks support (BP trees (nchuck ϭ 10, chuckscore ϭ 1) per iteration. The number of RAS took Ͻ 50; PP 0.68). Euphorbioideae are not monophyletic due to into account the number of times the shortest tree was found during the ®rst the pulling away of Stomatocalyceae which group as the un- stage of the heuristic searches (see Kauff and Lutzoni, 2002). Preliminary supported sister to the inaperturate crotonoids. The core aca- tests with 5±200 RAS also empirically veri®ed the threshold needed to hit the lyphoids and inaperturate crotonoid subclade C2 are also poor- same minimal length at least twice per bootstrap replicate. For rbcL and com- ly resolved. Among all nodes with PP 0.50±1.0, 65 (36%) had bined data sets, 100 RAS were used (shortest tree in heuristic search found PP 0.50±0.94 and fall below the signi®cance criteria noted an average of 1 per 100 RAS) and 20 RAS for trnL-F (shortest tree found earlier (Materials and Methods, Sequence Assembly and Data an average of 4 per 10 RAS). Bootstrap percentages (BP) are described as Analysis). high (85±100%), moderate (75±84%), or low (50±74%). Bayesian inference was performed with MrBayes 3.0b4 (Huelsenbeck and Analyses of trnL-FÐThe trnL intron ranged in length from Ronquist, 2001), and data were partitioned by gene in the combined analysis. 419 (Aubletiana) to 759 bp (Euphorbia epithymoides), but The most appropriate model for each paritition was determined with Modeltest most accessions fell between 500 and 545 bp. The trnL-F in- v3.06 (Posada and Crandall, 1998) to be the general time reversible model ϩ ϩ⌫ tergenic spacer ranged from 135 (Adenocline pauci¯ora)to (GTR I ). Four Markov chain Monte Carlo (MCMC) chains (one cold 476 bp (Julocroton), but most were 370±440 bp. Large dele- and three heated with default heating parameter of 0.2, and each initiated with Ͼ ϫ 7 tions in the spacer ( 100 aligned bp) occurred sporadically a random tree) were run simultaneously for 1 10 generations and sampled with at least nine independent major events. Among these was every 100 generations. We used default priors and a Dirichlet distribution for the base frequency parameters. Results (i.e., Bayesian trace ®les with sampled a large synapomorphic deletion of 226 bp (relative to aligned parameter values) were examined with Tracer v1.0.1 (Rambaut and Drum- length; 103 bp inferred absolute length compared with sister mond, 2002±2003) to determine when stationarity had been reached. The group, subclade C1) uniting some members of six tribes of stationary phases of two independent replicate runs were pooled after dis- inaperturate Crotonoideae (see Fig. 4, subclade C2). The trnL- carding trees for each replicate from the burn-in period that included the ®rst F matrix consisted of 220 sequences, and the aligned length 1.0 ϫ 106 generations (10 000 trees) for trnL-F and combined data sets and of 2298 sites was 1.7ϫ longer than the absolute length of the 1.5 ϫ 106 generations (15 000 trees) for rbcL. Posterior probabilities (PP) Ն longest component sequence. A homoplasious microinversion 0.95 were considered signi®cant (Kauff and Lutzoni, 2002). in the alchorneoids and subclade A1 was associated with a probable hairpin secondary structure (see Kelchner and Wen- RESULTS del, 1996). The MP strict consensus is shown in Fig. 2. Eu- phorbiaceae s.s. are strongly supported, and the spine of the Data set characteristics and search statistics are presented in tree is better resolved than with rbcL; all nine major lineages Table 1. The parsimony and Bayesian analyses exhibited a are recovered intact, but the core acalyphoids and inaperturate general correspondence and did not have supported incongru- crotonoid subclade C2 are poorly resolved. There were 41 ence; bootstrap-supported nodes mostly also had high PP. nodes (24% of nodes with PP Ն 0.50) with PP 0.50±0.94. Bayesian results were better resolved than MP strict consensus One notable topological difference was the placement of Man- trees when including nodes with PP Ͼ 0.50, but this difference niophyton ϩ Pausandra as sister to the rest of poorly resolved and most of the slight topological differences disappeared subclade C2; Trigonostemon occupies this position in the rbcL when considering nodes with PP Ն 0.95. and combined analyses. Euphorbiaceae s.s. and Pandaceae were recovered as strong- ly supported monophyletic groups. Of the three uniovulate CombinedÐThe combined data set had 179 terminals with subfamilies that comprise Euphorbiaceae s.s. in current clas- data for both partitions, and the MP analysis yielded 87 862 si®cations, only Euphorbioideae were here found to be mono- trees when searched to completion from seed trees collected phyletic. Acalyphoideae are clearly paraphyletic by containing from replicates of RAS in the ®rst stage of heuristic searches. the acalyphoid grade separate from the core remainder of Aca- The strict consensus is shown in Figs. 3 and 4. Support across lyphoideae and by the exclusion of Omphalea, which was the tree is higher than in the single locus analyses (inferred found embedded in a crotonoid lineage. Crotonoideae do not from the presented trees or more directly evident in partitioned form a supported monophyletic group but relationships among analyses of the combined data set, not shown). Euphorbiaceae the four main lineages are unclear. In total, nine major lineages s.s. are strongly supported, and all nine major lineages are (Acalyphoideae s.s., Adenoclineae s.l., articulated and inaper- recovered intact; inaperturate crotonoid subclade C2 is again turate crotonoids, Cheilosoideae, Erismantheae, Euphorbioi- poorly resolved. Bayesian analyses had 28 nodes (18% of deae, Gelonieae, and Peroideae) of Euphorbiaceae s.s. were nodes with PP Ն 0.50) with PP 0.50±0.94, including two to- August 2005] WURDACK ET AL.ÐEUPHORBIACEAE PHYLOGENETICS 1401

Fig. 1. Strict consensus of 1.0 ϫ 106 minimal length trees (tree length ϭ 2358, CI ϭ 0.306, RI ϭ 0.706) resulting from parsimony analysis of rbcL data for Euphorbiaceae s.s. The numbers above the branches are bootstrap percentages Ն50%; indicated below branches are posterior probabilites Ն0.95 in the Bayesian analyses. pological differences between the Bayesian and MP trees lished. Pandaceae are excluded here and are far removed from among the major lineages (i.e., Bayesian: Euphorbioideae ϩ the position among the Acalyphoideae suggested by Webster core acalyphoids; Erismantheae sister to articulated crotonoids (1994b). A sister relationship to Euphorbiaceae s.s. should not ϩ inaperturate crotonoids). The alternative placements (i.e., be inferred by close proximity in our trees because that posi- Erismantheae ϩ Euphorbioideae) of these groups shown in the tion merely re¯ects our limited sampling of families in Mal- MP tree lack bootstrap support (BP Ͻ 50). pighiales. Supported sister groups to both Pandaceae and Eu- The combined analysis represents the best overall picture of phorbiaceae s.s. remain to be established, although larger or- phylogenetic relationships in the family and the one on which dinal phylogenetic studies beyond the present four-gene study our discussion is focused, except if taxa were only sampled (Davis et al., 2005) are underway that may provide support for one of the single-gene analyses. for critical deep nodes (Wurdack and Davis, unpublished data). In our trees, no additional lineages were excluded from Eu- DISCUSSION phorbiaceae s.s.; morphologically aberrant Dicoelia is now The phylogenetic analyses presented here show broad cor- known to be a member of Phyllanthaceae (Kathriarachchi et respondence to recently proposed classi®cations that have not al., 2004, 2005). Centroplacus, of uncertain familial af®liation incorporated any information from DNA, the notable excep- (APG II, 2003) but usually associated with Phyllanthaceae or tions involving historically contentious or problematic groups. Pandaceae, has also been excluded (not sampled here; see Until now, delimitation of Euphorbiaceae s.s. has largely relied Wurdack et al., 2004). on inferences from previous sensu lato family classi®cations. The spines of the trees in all analyses lack supported rela- A robust circumscription of Euphorbiaceae s.s. is here estab- tionships (BP Ͻ 50) among major clades that relate to sub- 1402 AMERICAN JOURNAL OF BOTANY [Vol. 92

Fig. 2. Strict consensus of 1.0 ϫ 106 minimal length trees (tree length ϭ 2257, CI ϭ 0.425, RI ϭ 0.785) resulting from parsimony analysis of trnL-F data for Euphorbiaceae s.s. The numbers above the branches are bootstrap percentages Ն50%; indicated below branches are posterior probabilites Ն0.95 in the Bayesian analyses. familial delimitation sensu (Webster, 1994b), especially among grains, rubber and Frey-Wyssling particles, and lutoids), in- lineages of Crotonoideae. Crotonoid pollen and phorbol esters dicate a potential source of characters that have been little (co-carcinogenic diterpenes) present in part of the family have studied on a broad scale. Milky latex (rarely yellowish) char- been used to link Euphorbiaceae with Thymelaeaceae acterizes Euphorbioideae and articulated crotonoids; red latex (Schmidt, 1986; Webster, 1987; Nowicke, 1994; Seigler, is frequent in inaperturate crotonoids and Adenoclineae s.l. 1994). These characters are homoplasious between the families Recently documented observations show isolated occurrences (but informative within them), despite their remarkable co- of laticifers in Acalyphoideae (i.e., Dalechampia; Hayden and distribution and absence elsewhere in angiosperms; Thyme- Hayden, 2000) along with latex-like exudate in Macaranga. laeaceae are ®rmly established in Malvales (Soltis et al., 2000; Laticifers have also been rarely observed in Phyllanthaceae APG II, 2003). In Euphorbiaceae s.s., crotonoid pollen is con- (Phyllanthus, Balaji et al., 1996; Dicoelia, Hayden and Hay- ®ned to Crotonoideae and phorbol esters nearly exclusively den, 2000). Older reports (reviewed by Rudall, 1987) are dif- (but see subclade A3) to Crotonoideae ϩ Euphorbioideae, in- ®cult to evaluate and have often been dismissed due to lack dicating that both are derived within the family rather than of documentation and corroboration. They may re¯ect differ- plesiomorphic. ences in interpretation (i.e., they actually are sclereids) and The distributions of latex and associated laticifers have been dif®culty of observation when laticifers are few. Rudall (1994) used in the broad classi®cation of Euphorbiaceae s.l., notably has suggested putative homology between laticifers and foliar their presence in Crotonoideae s.l. and Euphorbioideae and sclereids. Foliar sclereids have been recently reported from absence in other lineages (i.e., Acalyphoideae, Phyllanthaceae, Chaetocarpus (Peroideae) and the core acalyphoid Mallotus and Picrodendraceae). Great differences in latex properties, (Roth, 1984; Rudall, 1994), as well as in some inaperturate such as color (e.g., milky, yellowish, orange, red, or clear), crotonoids (Rudall, 1994). A preliminary survey (K. J. Wur- chemical composition, and subcellular bodies (i.e., starch dack, unpublished data) of c. 600 Euphorbiaceae s.l. cleared August 2005] WURDACK ET AL.ÐEUPHORBIACEAE PHYLOGENETICS 1403

Fig. 3. Strict consensus of 87 862 minimal length trees (tree length ϭ 4288, CI ϭ 0.374, RI ϭ 0.711) resulting from parsimony analysis of combined rbcL and trnL-F data for Euphorbiaceae s.s. The numbers above the branches are bootstrap percentages Ն50%; indicated below branches are posterior probabilites Ն0.95 in the Bayesian analyses. Major clades are indicated and select characters mapped. Tribal classi®cation, biogeography, and petal status from Radcliffe- Smith (2001); seed vascular bundle status from Tokuoka and Tobe (1998, 2002, 2003). Tribal abbreviations: ACAL±Acalypheae, ADEL±Adelieae, ADEN± Adenoclineae, AGRO±Agrostistachydeae, ALCH±Alchorneeae, ALEU±Aleuritideae, AMPE±Ampereeae, BERN±Bernardieae, CARY±Caryodendreae, CHAE± Chaetocarpeae, CHEI±Cheiloseae, CHRO±Chrozophoreae, CLUT±Clutieae, CODI±Codiaeae, CROT±Crotoneae, ELAT±Elateriospermeae, EPIP±Epiprineae, ERIS±Erismantheae, EUPH±Euphorbieae, GALE±Galearieae, GELO±Gelonieae, HIPP±Hippomaneae, JATR±Jatropheae, MANI±Manihoteae, MICR± Micrandreae, OMPH±Omphaleae, PACH±Pachystromateae, PERE±Pereae, PLUK±Plukenetieae, POGO±Pogonophoreae, PYCN±Pycnocomeae, RICC± Ricinocarpeae, RICD±Ricinodendreae, STOM±Stomatocalyceae, TRIG±Trigonostemoneae. leaves deposited at the Smithsonian Institution (J. Wolfe col- gomuellera, Blumeodendron, Cleidion, Crotonogynopsis, Er- lection, Division of Paleobotany) revealed a far more complex ismantheae, Haematostemon, Koilodepas, Pera, and Pseuda- picture with wider sclereid distribution, intrageneric variation grostistachys) and crotonoids (Dodecastigma, Pausandra, and (i.e., present in some but not all species of Cleidion, Pera, and Trigonostemon). In addition to our poor knowledge of laticifer Trigonostemon), and intermediate states (perhaps weakly distribution (i.e., Gelonieae apparently remain to be investi- scleri®ed). Well-developed elongate sclereids were observed gated), the lack of support among major clades hinders un- in acalyphoids (Adenophaedra, Agrostistachys, Angostylis, Ar- covering the pattern(s) of laticifer evolution in the family. 1404 AMERICAN JOURNAL OF BOTANY [Vol. 92

Fig. 4. Continuation of Fig. 3 with strict consensus of parsimony combined analysis of rbcL and trnL-F data for Euphorbiaceae s.s. August 2005] WURDACK ET AL.ÐEUPHORBIACEAE PHYLOGENETICS 1405

Likewise, we have found no support for the widely held hy- atypical forms. Indehiscent fruits are clearly homoplasious and pothesis that articulated laticifers are derived from nonarticu- presumably associated with shifts to zoochory (e.g., Croton- lated ones. The former are multicellular and originate in pri- opsis, Elaeophorbia, Glycydendron, Mareyopsis, Ricinoden- mary and secondary tissues, whereas nonarticulated types are dron, and Trewia; see Esser, 2003b). Seed arils are of probable unicellular (coenocytic) and nearly exclusively originate in pri- phylogenetic utility, although their homologies are poorly mary tissues (Rudall, 1994). Articulated laticifers occur in two known, and they are notably scarce or absent in Acalyphoi- clades (Jatropha and articulated crotonoids) and have been deae, Adenoclineae s.l., and Gelonieae (see Tokuoka and Tobe, suggested to have two independent origins (Dehgan and Craig, 1998, 2002, 2003, but underestimated compared with Rad- 1978). Neither occurence is in a supported position within the cliffe-Smith, 2001). Most of these arils are con®ned to the nonarticulated clades. The presence of articulated laticifers in micropylar region (caruncles); they have been usually consid- ``basal'' lineages of Malpighiaceae has been used to suggest ered to be involved in dispersal (e.g., Berg, 1975; Narbona et their presence in the common ancestor of Euphorbiaceae s.s. al., 2005), but they also effect seed physiology (Webster, and Malpighiaceae (Vega et al., 2002), although there is no 1994a; Bianchini and Pacini, 1996). Caruncles vary greatly in support for a close relationship between these families. Elatin- morphology and distribution across the family, even within aceae, the apparent sister to Malpighiaceae, lacks laticifers, some genera (e.g., Croton, Euphorbia s.l., and Stillingia)or although some members possess a brownish resin (see Davis individual plants (i.e., seeds from allomorphic vs. regular ¯ow- and Chase, 2004). ers in Acalypha). Peroideae are carunculate, although Tokuoka Indument type and reproductive features including the pres- and Tobe (2003) described Pogonophora as exarillate. Our ob- ence of petals in pistillate and/or staminate ¯owers (mapped servations of herbarium specimens indicate that Pogonophora in Figs. 3, 4), pollen type (e.g., inaperturate crotonoid exines), has a ¯eshy and well-developed caruncle on mature, black and pollen nuclear number may be useful at higher levels. seeds, in agreement with other reports (Secco, 1990; Radcliffe- Trichome morphology varies greatly across the family and Smith, 2001). Caruncles are widespread in Picrodendraceae even within a genus (e.g., Croton s.l.; Webster et al., 1996), but notably lacking in Podocalyx, which is sister to the rest of but there appears to be broad utility in simple vs. stellate that family (K. J. Wurdack, unpublished data) and absent from forms; specialized types such as malpighian and stinging hairs Phyllanthacaeae except for Celianella (Stuppy, 1996). can be informative for some groups. Although pollen nuclear data are sparse, we estimate from comparisons of the data The acalyphoid gradeÐA series of mono- or digeneric tabulated by Webster and Rupert (1973) with our trees and for tribes that were all previously assigned to Acalyphoideae fall Euphorbioideae with those of Steinmann and Porter (2002) into two clades that are subsequent sisters to the rest of Eu- that the trinucleate condition has been gained at least eight phorbiaceae s.s.; the ®rst clade containing Chaetocarpeae, Clu- times. Cytological data show great variation but are sparse tieae, Pereae, and Pogonophoreae (Peroideae; see conclusions) except for the larger genera (Hans, 1973; Urbatsch et al., 1975; and the second with Cheiloseae (Cheilosoideae, see conclu- Index to Plant Chromosome Numbers, Missouri Botanical sion). These tribes were already considered primitive and Garden, St. Louis, Missouri, USA. Available at http://mobot. placed ®rst in Webster's (1994b) phylogenetic classi®cation mobot.org/W3T/Search/ipcn.html). The articulated crotonoids that started with Clutieae and ended in Euphorbieae. appear to have chromosome numbers based on x ϭ 9, whereas Peroideae is sister to all other Euphorbiaceae s.s. The fruit those of the core acalyphoids, Gelonieae, and most inapertur- morphology (membranous, fragile septa without visible vas- ate crotonoids and Euphorbioideae are based on x ϭ 11. Most cularization) of these taxa sets them apart from all other Eu- Euphorbia s.l. are derived from dysploid series of x ϭ 6±10. phorbiaceae s.l. (Esser, 2003a). They also share distinctive The alchorneoids and inaperturate crotonoid subclade C1 have smooth, shiny, black, carunculate seeds. Chaetocarpus, Clutia, lineages (Discoglypremna and Jatropha, respectively) based and Pera, each from separate tribes, form a strongly supported on x ϭ 11. subclade within Peroideae and have a unique exotestal seed A study of comparative seed and ovule anatomy has re- coat (Tokuoka and Tobe, 2003). The seed characters appear to cently been completed across Euphorbiaceae s.s. (Tokuoka and be apomorphic in the family, and their strongly supported sis- Tobe, 1998, 2002, 2003). The importance of these characters ter Pogonophora and the rest of Euphorbiaceae s.s. have ex- will depend on their evolutionary stability both within the fam- otegmic, palisadal seed coats (Tokuoka and Tobe, 2003; Po- ily and across other lineages of Malpighiales as indicated in gonophora recon®rmed here, K. J. Wurdack, personal obser- phylogenetic analyses such as ours; they need to be studied in vation). Pera has been considered isolated and classi®ed in its Malpighiales generally. Vascular bundles in the outer integu- own tribe or even family (see Radcliffe-Smith, 1987) because ment (mapped in Figs. 3, 4) are clearly homoplasious within of its unique pseudanthial in¯orescences. A survey of Pera Euphorbiaceae s.s. and also when compared to other families pollen noted high diversity, including an intectate form, de- (i.e., Phyllanthaceae; Tokuoka and Tobe, 2001; Wurdack et al., spite the general morphological similarity of most species 2004). The presence of vascular bundles in the inner integu- (Nowicke et al., 1998). Specialized pollinator relationships are ment appears more conservative (absent in Phyllanthaceae; expected to be involved with ¯oral novelties, but in the case Tokuoka and Tobe, 2001) and characteristic (a possible syna- of Pera they do not seem to be unusual; recorded visits are pomorphy) of most crotonoids (mapped in Figs. 3, 4). It is from small pollen-collecting bees (Gillespie and Armbruster, unclear how much of their distribution re¯ects plesiomorphic 1997). The strongly supported relationship of Pera with Aca- absence, secondary losses, or possible multiple gains, espe- lypha (Figs. 2, 4 in Davis and Chase, 2004) is an artifact of cially when considering their isolated occurence in Anthostem- sparse sampling relative to exceptionally high sequence diver- inae (Euphorbioideae) and Klaineanthus (Adenoclineae). As gence in PHYC (PHYC independently resequenced and veri- in Phyllanthaceae and Picrodendraceae, typical explosive eu- ®ed, K. J. Wurdack, unpublished data). The putative associa- phorbiaceous schizocarps are also predominant in Euphorbi- tion of Pera with Alchorneeae based on wood anatomy (Hay- aceae s.s., although most taxa in the acalyphoid grade have den and Hayden, 2000) is not supported here. Pera is Neo- 1406 AMERICAN JOURNAL OF BOTANY [Vol. 92 tropical, Clutia African (with greatest diversity in South cepsia (rbcL only), a member of Bernardieae (Webster, 1994b; Africa), and Pogonophora is vicariant (one Neotropical spe- Radcliffe-Smith, 2001), is strongly supported as sister to Pseu- cies and one West African). Chaetocarpus has a disjunct trop- dagrostistachys in the rbcL analysis and this pair is in turn the ical distribution, including species in the Neotropics, Africa, unsupported (BP Ͻ 50) sister to the core acalyphoids. Alter- and Asia. native rbcL analyses (not shown) without Paranecepsia re- The next node in the acalyphoid grade isolates Neoscorte- cover the placement of Pseudagrostistachys sister to the al- chinia (Cheilosoideae) from the rest, although this node is chorneoids (BP 68) seen in the trnL-F and combined trees. poorly supported (BP 72). Cheilosoideae comprise two small The placement of Amyrea differs greatly between Webster Southeast Asian genera that are palynologically unique in Eu- (1994b; Acalypheae-Claoxylinae) and Radcliffe-Smith (2001; phorbiaceae s.s. due to their echinate pollen exines (Takahashi Bernardieae). Radcliffe-Smith (1998) noted its morphological et al., 1995; Nowicke et al., 1998). Homoplasious echinate similarities with Agrostistachys ϩ Pseudagrostistachys, and exines are present in other euphorbiaceous lineages, including this af®nity is supported here. Alchorneeae s.s. (sensu Webster, Amanoa and Croizatia of Phyllanthaceae, and most Picroden- 1994b) form a strongly supported clade within the alchor- draceae. Ovule and seed characters also support the distinc- neoids and have potential synapomorphies in wood anatomy tiveness of Cheilosoideae (Tokuoka and Tobe, 2003). (lysigenous canals; Hayden and Hayden, 2000) and pollen (strati®ed opercula; Nowicke and Takahashi, 2002). The nest- Core Euphorbiaceae s.s.ÐThe rest of the family forms a ed nature and supported nodes of sister taxa indicate that Al- monophyletic group containing seven major lineages: (1) Er- chorneeae s.s. might be expanded (i.e., Alchorneeae s.l.) to ismantheae, (2) Acalyphoideae s.s., (3) Adenoclineae s.l. (in- include some or all members of the alchorneoid clade. Outside cluding Omphalea but excluding Glycydendron), (4) Gelon- Alchorneeae s.s. there is no great morphological discontinuity ieae, (5) articulated crotonoids, (6) inaperturate crotonoids, and along this grade; however, Pseudagrostistachys and its pre- (7) Euphorbioideae (including Stomatocalyceae). sumed sister Agrostistachys (not sampled) have petals in ¯ow- ers of both sexes, and Cyttaranthus has petals only in stami- ErismantheaeÐThe next tribe after Cheiloseae in Webster's nate ¯owers. Petals are absent in the remaining alchorneoids linear sequence is Erismantheae. This small Southeast Asian and Paranecepsia. The position in our analyses of Mareyopsis group of three genera, totaling only ®ve species, is united by among the alchorneoids is far removed from Mareya (see core monoecy, opposite leaves, and interpetiolar stipules. The un- acalyphoid subclade A3) despite recent synonymization by usual stipular arrangement has been suggested to be the result Webster (1994b). Radcliffe-Smith (2001) reinstated the genus of unequal internode growth (i.e., seemingly opposite leaves as distinct from Mareya but placed both in a newly recognized due to contracted internodes) and loss of one stipule per leaf subtribe Mareyinae within Acalypheae. The heterogeneous (Welzen, 1995; Radcliffe-Smith, 2001). Opposite leaves have pollen observed (Nowicke and Takahashi, 2002) in Mareyop- evolved independently multiple times across the family (e.g., sis is based on misidenti®cation (Zenker ``35'' incorrect, K. J. Baloghia inophylla, Calycopeplus spp., Chamaesyce spp., Wurdack, personal observation). Trigeneric Caryodendreae Mallotus section Hancea, Mercurialis spp., Pera oppositifolia, dissolve with the placement of Discoglypremna and Alchor- Sebastiania hexaptera, and Stillingia oppositifolia), but inter- neopsis as separate alchorneoid lineages and Caryodendron in petiolar stipules occur only in Erismantheae and, deeply em- subclade A7. African Discoglypremna and Neotropical Al- bedded in the euphorbioid clade, Chamaesyce. The sampled chorneopsis are distinct as indicated by seed coat data (Tok- Erismantheae, Moultonianthus (rbcL only) and Syndyophyllum uoka and Tobe, 2003) and not vicariants as suggested by Web- form a monophyletic group in the rbcL analyses. The last, ster (1994b). Our data indicate that Conceveiba sensu Webster sampled for the combined analyses, is resolved among the lac- (1994b) and Radcliffe-Smith (2001) is not monophyletic and tiferous lineages, but this position lacks support (BP Ͻ 50). support the separation of Aubletiana, containing two African Laticifers have not been reported in the tribe (Hayden and taxa originally described as Conceveiba species, from the Neo- Hayden, 2000), but the leaves are scleri®ed (Herbert, 1897; K. tropical remainder (Murillo-A., 2000). The position of Gavar- J. Wurdack, personal observation). retia is unresolved, but its inclusion along with Polyandra (not sampled) in Conceveiba s.l. has been proposed (Murillo-A., Acalyphoideae s.s.ÐAcalyphoideae s.s. form a strongly 2000) and is supported in a better-sampled nuclear ITS ribo- supported clade uniting the remaining 11 tribes (excluding, as somal DNA analysis (K. J. Wurdack, unpublished data). Pan- noted, Dicoelieae and Galearieae) of Acalyphoideae sensu tropical Alchornea is paraphyletic with the inclusion of Boc- Radcliffe-Smith (2001). The clade contains two strongly sup- quillonia, a small genus endemic to New Caledonia. The spell- ported groupings with the alchorneoids sister to the ``core aca- ing ``Alchorneeae'' is here adopted in accordance with the lyphoid'' remainder. The core acalyphoids are poorly support- nomenclatural code (Greuter et al., 2000) Art. 19.3. and 18.1. ed at the deepest nodes, but do contain some supported internal The spelling ``Alchorneae'' (Webster, 1994b) or ``Alchor- subclades (designated for discussion subclades A1±A7) that, nieae'' (Radcliffe-Smith, 2004) contravenes these rules. The in part, follow previous tribal or subtribal classi®cations. same applies to ``Ampereeae,'' not Ampereae (Webster, 1994b; Radcliffe-Smith, 2001). Both Alchornea and Amperea are AlchorneoidsÐOne of the two constituent main groupings based on the surname of a person. of Acalyphoideae s.s. is a novel association of Alchorneeae ϩ Agrostistachydeae and several genera from three other tribes Core acalyphoid subclade A1ÐBlumeodendron (Pycnoco- including Caryodendreae, which Webster (1994b) suggested meae±Blumeodendrinae) is strongly supported as sister to Ma- had links to Agrostistachydeae. Pseudagrostistachys is sister caranga, Mallotus, and Trewia (Acalypheae±Rottlerinae). to the rest of this subclade in the combined analyses (but not Webster's (1994b) doubts about the monophyly of Pycnoco- rbcL), making Agrostistachydeae paraphyletic by not grouping meae are con®rmed by our analyses, with all sampled mem- with the other sampled tribal member, Cyttaranthus. Parane- bers of Pycnocomeae±Pycnocominae (Argomuellera and Pyc- August 2005] WURDACK ET AL.ÐEUPHORBIACEAE PHYLOGENETICS 1407 nocoma) far removed to subclade A3. Blumeodendron has a lypheae±Mareyinae by Radcliffe-Smith (2001), but the ®rst disc in ¯owers of both sexes; this is absent in Macaranga and species were described by Bentham in Acalypha (Hooker, Trewia but variably present in Mallotus (disc glands 0±ϱ ac- 1849; see also Bentham, 1878). Both genera share dissected cording to Radcliffe-Smith, 2001). Blumeodendron shares the styles, divergent anther thecae, and similar pollen exines stellate indumentum with Macaranga, Mallotus, and Trewia, (Nowicke and Takahashi, 2002). Mareya does not appear to whereas Pycnocominae have simple hairs. Mallotus is sister to have the full suite of anemophilous adaptations as it possesses Trewia in our analyses, and this strongly supported clade is in interstaminal glandular disc segments and lacks the pollen re- turn sister to Macaranga. Mallotus japonicus (section Mallo- duction (i.e., smaller size and shorter colpi) of Acalypha. Cro- tus) and M. philippensis (section Rottlera; trnL-F only) in our tonogynopsis is currently classi®ed in tribe Adelieae but was sampling are embedded in the genus based on morphological placed next to Mareya by Pax and Hoffmann (1931). This analyses (Slik and Welzen, 2001; although most relationships ditypic African genus seems to be wind-pollinated with long in their tree lack strong support and Trewia was not included pendulous, sometimes cauline staminate racemes and long sta- there), indicating that Trewia may also be embedded within mens protruding from the calyx. The basally divergent anther Mallotus. The only character used to distinguish Mallotus and thecae and laciniate styles provide another link with the de- Trewia is fruit type (dehiscent and indehiscent, respectively). rived features seen in Acalypha. Transitions between these have been shown to occur frequently The last strongly supported grouping in subclade A3 in- within Euphorbiaceae s.l. (see Wurdack et al., 2004) and in cludes Pycnocoma ϩ Argomuellera (Pycnocomeae±Pycno- the case of Trewia may be adaptive for megafaunal dispersal cominae) and Sampantaea (trnL-F spacer only) ϩ Wetria. The (Dinerstein and Wemmer, 1988). The granular laminar glands latter pair was considered closely related and perhaps conge- characteristic of Macaranga and Mallotus pro parte are also neric (Webster, 1994b), although they differ in pollen charac- present on young leaves of Trewia (P. Hoffmann, personal ob- ters (Takahashi et al., 2000). Palynologically, Sampantaea is servation; not noted in Radcliffe-Smith, 2001). Webster close to Cleidion, but the latter is phylogenetically distant in (1994b) considered the eight genera of Acalypheae±Rottleri- our analyses (subclade A1). Wetria and Pycnocoma are anom- nae (only two Mallotus species and monotypic Trewia sam- alous among Acalyphoideae s.l. in having bioactivity for phor- pled here) to be closely related, but further sampling within bol esters, but this was not detected in Sampantaea (Argo- the subtribe and aberrant sections of Mallotus (i.e., Hancea muellera not examined; Beutler et al., 1996). Wood anatomy and Oliganthae) is needed to determine how to treat Mallotus. has been used to support an alignment of Wetria with Al- Subclade A1 is nearly exclusively Old World, centered in trop- chorneeae (i.e., anomalously large rays that might be pre-ly- ical Asia. Exceptions are in Cleidion, the poorly supported sigenous; Hayden and Hayden, 2000), but this disposition is sister to subclade A1, with three of c. 25 species from the also not supported palynologically nor by our results. Neotropics (including C. castaneifolium sampled here). Rad- cliffe-Smith (2001) also included the poorly known Chilean Core acalyphoid subclade A4ÐSubclade A4 is a convenient endemic Avellanita (not sampled) in Rottlerinae, but an as- unit for discussion but poorly supported (BP 62) compared sociation with the members of principally American subclade with the other acalyphoid subclades recognized here. The sub- A5 has also been suggested (see Webster, 1994b). clade is exclusively Old World and contains several groups recognized in previous classi®cations including Ampereeae, Core acalyphoid subclade A2ÐLobanilia (Acalypheae±Lo- Epiprineae, and three of four subtribes of heterogeneous Chro- baniliinae) is embedded in Acalypheae±Claoxylinae in our zophoreae. Ricinus is here and previously has occupied an iso- analyses. The small Madagascan genus, formerly a section of lated position in the monotypic Acalypheae±Ricininae, al- Claoxylon, has been segregated as Acalypheae±Lobaniliinae though Webster (1994b) suggested an af®liation with Adriana. because of its stellate indumentum and lack of reddish pigment The latter genus associates with Monotaxis (and Amperea for (Radcliffe-Smith, 1989), but is otherwise similar to the other rbcL only) in our trees to form a strongly supported exclu- taxa in this morphologically homogeneous group. Sister to this sively Australian grouping sister to the remainder of subclade clade is Mercurialis, placed far from the two other members A4. Our analyses uncover new associates for Ricinus including of Acalypheae±Mercurialinae (subclade A5). Mercurialinae is Speranskia, poorly supported as its sister. Both genera differ heterogeneous geographically (Eurasia vs. southern Africa) in many ways, notably in the numerous fascicled stamens of and with respect to breeding system, stamen number, as well Ricinus vs. 10±15 free stamens in Speranskia. Fascicled sta- as disc and seed morphology. mens are homoplasious in Acalypheae±Lasiococcinae, includ- ing Homonoia (rbcL only) and Spathiostemon sampled here Core acalyphoid subclade A3ÐA clade uniting Spathioste- (subclade A3). In addition to Ricinus, many members of sub- mon and (rbcL only) Homonoia (Acalypheae±Lasiococcinae) clade A4 contain homologous genes for the proteinaceous is sister to two strongly supported groupings, one containing seed-toxin ricin (K. J. Wurdack, unpublished data). Suregada Acalypha and two new associates, Crotonogynopsis (rbcL (gelonin; Hosur et al., 1995) and Jatropha (curcin; Lin et al., only) and Mareya, and the other grouping with mostly Pyc- 2003) produce functionally similar ribosome inactivating pro- nocomeae±Pycnocominae. Acalypha, the third largest genus of teins (RIPs) but those taxa are unrelated in our analyses. Euphorbiaceae s.s. with 462 species (Govaerts et al., 2000), is distinctive by virtue of a suite of wind pollination adaptations Core acalyphoid subclades A5, A6ÐAn herbaceous habit is including small porate pollen, pendulous, ¯exuous to vermi- uncommon in Euphorbiaceae s.s., and four relatively delicate form anther thecae, reduced perianth, dissected styles, and lack genera (Dysopsis, Leidesia, Mercurialis, and Seidelia, all sam- of ¯oral nectar. As a consequence, it was placed in a mono- pled here) have been classi®ed in adjacent Acalypheae sub- generic subtribe Acalyphinae. Our results show Acalypha as tribes Mercurialinae and Dysopsidinae. Southern African Lei- the strongly supported sister of Mareya. Mareya was placed desia and Seidelia form a supported monophyletic group (A5) in Acalypheae±Claoxylinae by Webster (1994b) and in Aca- as expected, given the suggestion by Webster (1994b) to unite 1408 AMERICAN JOURNAL OF BOTANY [Vol. 92 them. Tokuoka and Tobe (2003) overemphasized the caruncle Gillespie (1994a), in evaluating pollen evidence, suggested in Seidelia (absent in Leidesia) as indication of misplacement; that Dalechampia with tricolporate pollen was sister to the the other two genera are not related (A2, Mercurialis; A6, remainder of the Plukenetieae rather than derived within it. In Dysopsis). Members of the strongly supported subclade A6 our analyses the position of Dalechampia (ϩ Astrococcus)is include Adelieae as sister to Chrozophoreae±Ditaxinae plus poorly supported as embedded. Tricolporate pollen is present Dysopsis (Acalypheae±Dysopsidinae). This clade is morpho- in most other Acalyphoideae (Nowicke and Takahashi, 2002), logically heterogeneous but many members have petals, and it whereas tricolpate apertures with uneven margins are a syna- is distributed nearly exclusively in the New World (except a pomorphy for the remaining members of the tribe (i.e., sub- few species of Caperonia). Relationships for Ditaxinae differ tribes Plukenetiinae ϩ Tragiinae). The tribe has a tendency greatly from those recovered in a morphological cladistic anal- toward aperture loss, but a reversal to the ancestral tricolporate ysis (Welzen, 1999), but the latter results show weak support condition is unlikely. Dalechampia is among the most spe- throughout as well as sampling constrained to follow Webster cialized genera of Euphorbiaceae s.s. in possessing a twining (1994b). Dysopsis, a genus of small Neotropical montane habit, stinging hairs, unusual pollen (Nowicke and Takahashi, herbs, is sister to also herbaceous Caperonia. Dysopsis had 2002), and unique, often colorful pseudanthia, for which evo- been included in Acalypheae±Dysopsidinae, associated with lution appears to be driven by pollinator relationships (Arm- other herbaceous taxa but is here recovered as sister to Cap- bruster and Baldwin, 1998). The perceived isolated nature of eronia which was classi®ed in Chrozophoreae. Pollen evi- the genus, based on derived species with highly modi®ed res- dence, in part from misinterpretation (see Takahashi et al., in-secreting pseudanthia, fails to take into account plesio- 2000), has been used to suggest an isolated position for Dy- morphic members in section Rhopalostylis (revised by Arm- sopsis (FernaÂndez-GonzaÂlez et al., 1994; Webster, 1994b). Di- bruster, 1996). Our analyses recover a strongly supported sister taxis and Chiropetalum, sisters in our analyses, have been con- relationship of Dalechampia and Astrococcus, thereby making sidered questionable segregates of Argythamnia s.l., although subtribe Plukenetiinae paraphyletic. Plukenetiinae have been ¯orally and palynologically they are distinct (Ingram, 1979). generally accepted as a natural group (Gillespie, 1994a; Web- Argythamnia s.s. was not sampled. A grouping of Adelia, La- ster, 1994b), although the three small genera (Angostylis, As- siocroton, and Leucocroton is strongly supported as had been trococcus, and Haematostemon) placed in the informal group suggested by pollen evidence (Takahashi et al., 2000) and ``Astrococciformes'' (Pax and Hoffmann, 1919) differ in habit shared tribal classi®cation in Adelieae. The remaining two (trees or shrubs instead of vines, but note secondarily shrubby members of that tribe, Crotonogynopsis and Enriquebeltrania, taxa in Acidoton s.l. and some Dalechampia), in¯orescence belong elsewhere (subclade A3 and unresolved in core aca- structure, ¯owers, and pollen from the rest of the subtribe and lyphoids, respectively) based on rbcL. Enriquebeltrania,a tribe (Gillespie, 1994a; Nowicke and Takahashi, 2002). Some monotypic southern Mexican endemic, had been considered members of Dalechampia are vines with stems becoming by Webster (1994b) to be questionably distinct from Adelia; woody with age (Armbruster, 1996, 2002; Armbruster and its position in the core acalyphoids is unresolved among the Baldwin, 1998). Gillespie (1994b, p. 330) noted ``Tragia as major subclades but clearly far removed from Adelia. presently circumscribed is distinguished from other members of subtribe Tragiinae by a combination of plesiomorphic char- Core acalyphoid subclades A7, A8ÐSubclade A7 contain- acter states.'' Tragia is paraphyletic in our better-sampled ing Caryodendron and two of three sampled members of Ber- trnL-F analyses, and the three sampled species each belong to nardieae is strongly supported as sister to subclade A8, the different sections. The distinctive section Bia represented by monophyletic Plukenetieae. Preoccupation with the great di- Tragia fallax is not sister to the others and has been previously versity of pollen and stylar morphologies in Plukenetieae has recognized at generic level (see Gillespie, 1994a, b). Addi- obscured understanding of phylogenetic relationships within tional sampling, especially of Tragiinae, with more rapidly this group despite probable synapomorphies such as twining evolving genes (e.g., nuclear ITS) is needed to allow a reclas- habit, pachychalazy (Tokuoka and Tobe, 2003), and stinging si®cation of Plukenetieae. hairs. Cnidoscolus (articulated crotonoids) has clearly inde- pendently evolved distinct stinging hairs (Solereder, 1908; Crotonoideae s.l.ÐSubfamily Crotonoideae, traditionally Webster, 1994a). Among the three Plukenetieae subtribes, circumscribed to contain lactiferous taxa with crotonoid pol- stinging hairs are present in Tragiinae and Dalechampinae but len, do not form a supported clade in our analyses but rather not Plukenetiinae. Most Plukenetiinae do, however, possess are divided into four clades including: (1) Adenoclineae s.l., potentially homologous ``Drusenhaare'' (Ritterhausen, 1892; (2) Gelonieae, (3) articulated crotonoids, and (4) inaperturate Solereder, 1908; K. J. Wurdack, personal observation) that also crotonoids. Corresponding to the ®rst dichotomy of Webster's occur independently in Acalypha (Cardiel, 1995) and Claox- (1994b) Crotonoideae tribal key, the Adenoclineae s.l., Gelon- ylon (Ritterhausen, 1892; not con®rmed here and see Soler- ieae, and articulated crotonoids share aperturate pollen and eder, 1908). These are typically stellate epidermal protuber- apetalous ¯owers, whereas the inaperturate crotonoids have ances formed by sur®cial foliar crystals, which in Plukeneti- petals and inaperturate pollen. Our combined tree does not inae could represent an intermediate state in the evolution (or unite the three aperturate-apetalous clades but indicates a poor- loss) of stinging hairs. The subepidermal initiation of stinging ly supported sister relationship between the articulated and the hairs in Tragia (Plukenetieae±Tragiinae) reported by Thurston inaperturate clades. A potential morphological synapomorphy (1976) indicates their derivation from foliar druses. The on- (mapped in Figs. 3, 4) for this articulated-inaperturate group, togeny of Plukenetiinae Drusenhaare and any lateral cell spe- including previously misplaced Elateriospermum and Glycy- cialization have not been studied, but they do appear to be dendron (see Adenoclineae s.l.), is seeds with thick inner in- subepidermally rooted (Ritterhausen, 1892, Figs. 5, 6; redrawn teguments that contain vascular bundles (Tokuoka and Tobe, in Solereder, 1908) as seen in early stages of Tragia emer- 1998). This diagnostic character also supports the exclusion of gence development (Thurston, 1976). Gelonieae and Adenoclineae (except Klaineanthus), which August 2005] WURDACK ET AL.ÐEUPHORBIACEAE PHYLOGENETICS 1409 have thin inner integuments lacking vascular bundles. A mor- ium are con®rmed here with a sister relationship to Suregada, phological and palynological cladistic analysis that primarily although a morphological and palynological cladistic analysis sampled from the articulated and the inaperturate clades bears placed them far apart (Lobreau-Callen et al., 2001). Suregada little resemblance to our topologies (Lobreau-Callen et al., and Clutia (Peroideae) are both characterized by leaves with 2001). unusual secretory lacunae (Solereder, 1908), giving them a pellucid-punctate appearance. This would appear to be hom- Adenoclineae s.l.ÐAdenoclineae sensu Webster contain six oplasious, given their distant placement in our results, and may genera (Adenocline, Ditta, Endospermum, Glycydendron, be adaptive for xeric environments. The lacunae seem to be Klaineanthus, and Tetrorchidium; all sampled here) that share missing in Cladogelonium, although this remains to be ex- aperturate pollen and apetalous ¯owers with the articulated amined for vestiges in its reduced, ephemeral leaves. As in crotonoids. The clade recovered in our analyses, although some Adenoclineae s.l., Suregada species are uraniine moth poorly supported, indicates that adjustments to this polyphy- host plants (Lees and Smith, 1991) and also contain AGIs (G. letic tribe are needed by excluding Glycydendron (see articu- Kite et al., Royal Botanic Gardens, Kew, unpublished manu- lated crotonoids) and including Omphalea. Possible synapo- script), which may indicate closer phylogenetic relationships. morphies are pollen exhibiting a similar acetolysis reaction (Nowicke and Takahashi, 2002) and the absence (except in Articulated crotonoidsÐArticulated laticifers and trinucle- Klaineanthus) of vascular bundles in their inner integuments ate pollen may be synapomorphic for the articulated croto- (Tokuoka and Tobe, 2002). Herbaceous Adenocline and arbo- noids, but these have only been documented in Cnidoscolus, rescent Klaineanthus are strongly supported sisters in our anal- Hevea, and Manihot (Hans, 1973; Webster and Rupert, 1973; yses despite disparate habits and seed coat anatomies. This pair Rudall, 1987, 1994); Elateriospermum and Glycydendron re- is in turn sister to a strongly supported subclade containing main to be investigated. Micrandra contains nonarticulated la- Ditta, Endospermum, Omphalea, and Tetrorchidium. Webster ticifers, but instances of anomalous cross-walls have been re- (1975, 1994b) classi®ed Omphalea with Acalyphoideae in the ported (Rudall, 1987, 1994). This clade unites three tribes monogeneric Omphaleae and placed it next to Plukenetieae (Manihoteae, Micrandreae, and Elateriospermeae) and in- based on shared palynological features and vining habit (Punt, cludes Glycydendron (Adenoclineae sensu Webster). Molecu- 1962; Gillespie, 1988; Nowicke and Takahashi, 2002). He also lar data place Elateriospermum here, as did earlier classi®ca- noted af®nities with Euphorbioideae and believed that ``clar- tions (see Webster, 1994b). Webster (1994b, p. 102) considered i®cation of its af®nities appears to be critical in establishing the genus ``a relict without close af®nities, but perhaps it is a the phylogeny of the uniovulate Euphorbiaceae'' (Webster, distant relative of Micrandra and Manihot.'' Noting its ina- 1994b, p. 96). The presence of abundant laticifers and latex in perturate pollen, however, he placed the monotypic Elaterios- Omphalea has been taken as an indication for misplacement permeae next to the inaperturate tribes. The well-developed of the genus in Acalyphoideae (Rudall, 1994). Omphalea and foot-layer and ``possible'' apertures observed by Nowicke Endospermum both (potential synapomorphy) have alkaloidal (1994) indicate that Elateriospermum does not belong with the glycosidase inhibitors (AGIs) and are host plants for specialist inaperturate crotonoids, as does the lack of petals. Southeast diurnal uraniine moths that accumulate these bioactive com- Asian Elateriospermum is anomalous in an otherwise strictly pounds (Kite et al., 1991). The distribution of AGIs in Eu- New World clade. Glycydendron is the poorly supported sister phorbiaceae s.s. remains to be fully surveyed but they are also to Hevea in our combined tree. Glycydendron is morphologi- present in Tetrorchidium (G. Kite, Royal Botanic Gardens, cally more similar to Micrandra (Micrandrinae; simple leaves, Kew, personal communication) and are expected in other free stamens) than Hevea (Heveinae; compound leaves, con- members of Adenoclineae s.l. and Gelonieae (see Gelonieae). nate stamens) but presents other features unique in the artic- The fused caplike androecium in Omphalea has been consid- ulated crotonoids, such as high stamen number and drupaceous ered unique, but it is part of an array of ®lament elaborations fruit. that characterize the clade. Similarly, there is style variation with a tendency toward short thickened styles (some caplike) Inaperturate crotonoidsÐThe inaperturate crotonoids are a in Adenoclineae s.l. Tetrorchidium is paraphyletic with Afri- well-supported clade uniting the remaining seven tribes of can T. gaboneae sister to a Neotropical clade that also contains Crotonoideae and sharing distinctive inaperturate ``crotonoid'' Ditta. The recognition of Hasskarlia to accommodate the Old pollen and petals at least in staminate ¯owers (except in apet- World taxa is supported by a number of morphological char- alous Benoistia and Neoboutonia). The pollen has an incom- acters (see Radcliffe-Smith, 2001). The exact relationship of plete tectum with ornamented, triangular supratectal elements Ditta with respect to New World Tetrorchidium s.s., however, of great variation, and a reduced endexine and foot layer to needs to be clari®ed. Although Ditta appears morphologically compensate for the lack of apertures (Nowicke, 1994). Our divergent from Tetrorchidium s.s. (more so than Hasskarlia), data resolve two strongly supported subclades (C1 and C2), it is from an area (Greater Antilles) of high endemism and for which the lack of apparent morphological synapomorphies morphological specialization, and further sampling is needed makes it impossible to even provisionally assign many of the to determine if it is sister to rather than embedded within Neo- unsampled genera, although there appear to be clear biogeo- tropical Tetrorchidium. graphic trends.

GelonieaeÐSuregada has porate pollen but an exine struc- Crotonoid subclade C1ÐSubclade C1 unites members of ture characteristic of inaperturate crotonoids (Nowicke, 1994); four tribes (Aleuritideae, Jatropheae, Codiaeae, and Crotoneae) it would appear to link the two palynological groups of Cro- with part of poorly sampled Jatropheae (Jatropha and Joan- tonoideae. Our analyses place it as sister to one or more of nesia) sister to the remainder. Subclade C1 is predominantly the crotonoid lineages, but those relationships lack support (BP distributed in the New World, and of the sampled genera only Ͻ 50). The af®nities of monotypic Madagascan Cladogelon- Croton and Jatropha have Old World radiations. The putative 1410 AMERICAN JOURNAL OF BOTANY [Vol. 92 plesiomorphic Jatropha section Curcas includes both Old and xylary phloem otherwise present in Croton s.l. (Solereder, New World members (Dehgan and Webster, 1979). Disposition 1908). The chromosome numbers in the rest of Croton s.l. are of the remaining six small genera of Jatropheae needs to be mostly derived from x ϭ 10 or 14 (Hans, 1973; Urbatsch et fully clari®ed, as their Old World distributions (except Vau- al., 1975). The recent generic recognition of Colobocarpos to pesia) and potential placement at basal nodes could affect the accomodate an aberrant Thai species of Croton appears un- interpretation of the biogeography of the subclade (i.e., New warranted. In our rbcL trees, it is embedded in Croton s.l. or Old World origin). African Leeuwenbergia is among these Brasiliocroton is sister to Croton s.l. excluding section Astraea critical Jatropheae genera and based on a partial rbcL sequence and is currently being described as a monotypic new genus falls in subclade C2; it is probable that some of the remaining that is distinctive in in¯orescence structure and in having erect genera have a similar disposition. Neotropical Vaupesia was anthers (Berry et al., in press). Webster (1994b) provisionally suggested by Webster (1994b, p. 104) to perhaps ``represent a included three other genera in Crotoneae: Fahrenheitia connecting link between tribes Micrandreae and Jatropheae'' (ϭ Paracroton), Mildbraedia, and Moacroton. Moacroton is (i.e., articulated vs. inaperturate crotonoids here). He ultimate- embedded in Croton s.l. Sampling of Mildbraedia (trnL-F ly preferred the inaperturate Jatropheae based on palynological only) and Paracroton has shown those genera to be misplaced; evidence (not seen by Nowicke, 1994, nor apparently ever they belong in the other inaperturate crotonoid subclade C2. critically examined by electron microscopy). The in¯orescence In our analyses, new associates of Croton are Neotropical structure and presence of petals and red latex (contrary to Ophellantha (ϭ Acidocroton sensu Webster), Sagotia, and white latex implied by Webster, 1994b) have all been noted Sandwithia, which all were of doubtful position in Webster's by Schultes (1955) to be similar to Joannesia, which would (1994b) system. Blachia had been linked to this subclade as suggest ties to Jatropheae. A trnL-F spacer sequence obtained the Asian vicariant of Acidocroton by Webster (1994a, b), but during manuscript revisions provided placement of Vaupesia the former belongs in subclade C2. Sagotia and Sandwithia as sister to Joannesia (analysis not shown). have been suggested to be related (Secco, 1988) as shown in The remaining members of subclade C1 (excluding Jatro- our analyses, but Webster (1994b) subsequently classi®ed pheae) could form the basis of an enlarged, revised Crotoneae. them in different tribes of Crotonoideae (Codiaeae and Aleu- The group contains Croton s.l., the largest genus of Crotono- ritideae, respectively). ideae (second largest in Euphorbiaceae s.s. after Euphorbia) with 1223 species recognized (Govaerts et al., 2000). The ge- Crotonoid subclade C2ÐThis subclade unites members of nus has been distinguished by anthers that are in¯exed in bud seven tribes and is supported by a large synapomorphic trnL- (i.e., the basi®xed anther and the apical portion of its ®lament F spacer deletion (Fig. 4, see Results, Analyses of trnL-F). are inverted, and righted at anthesis when the stamen is ex- Resolution is poor, and there are few strongly supported re- tended). Erect anthers have, in part, been used in the generic lationships, suggesting that more rapidly evolving genes are recognition of Colobocarpos and Moacroton for aberrant spe- needed. Trigonostemon is the poorly supported (rbcL)orun- cies formerly in Croton, but in our analyses both segregates supported (combined) sister to this subclade; trnL-F shows a are resolved within in¯exed Croton s.l. The type of Croton different unsupported sister group (Pausandra ϩ Mannioph- colobocarpus (Kerr 8479, K) has typically in¯exed anthers in yton). Although maintaining the monogeneric Trigonostemo- bud (P. Hoffmann, personal observation). Stamens of Moac- neae, Webster (1994b) has questioned its tribal distinctiveness. roton have nearly sessile anthers in the very small ¯owers, so Welzen and Stuppy's (1999) morphologically based analysis that it is physically impossible for them to be in¯exed at any using nearly exclusive (except Sandwithia) sampling within stage of their development. The character of anther position subclade C2 shows little correspondence to our results. Rela- needs to be critically examined, both developmentally and tionships between the Australian endemic genera Bertya, Be- with regards to its taxonomic distribution. Recorded observa- yeria, and Ricinocarpos on the one hand and between the Aus- tions are few and the morphological diversity of Croton s.l. is tralasian (both with taxa in New Caledonia) Baloghia and vast. Fontainea are strongly supported. Subclade C2 has a Paleo- Increased trnL-F sampling places the segregates Crotonop- tropical distribution in contrast to predominantly Neotropical sis, Eremocarpus, and Julocroton ®rmly within Croton as fa- subclade C1. Of the 46 genera potentially in subclade C2 (i.e., vored in the sensu lato circumscription of Webster (1993, the 28 sampled genera and their unsampled associates accord- 1994b). Radcliffe-Smith (2001) was retrograde in returning to ing to Radcliffe-Smith, 2001), only Anomalocalyx (not sam- the sensu stricto delimitation that makes Croton paraphyletic pled), Dodecastigma, Garcia, and Pausandra are Neotropical. in a fashion typical of large groups (e.g., Euphorbia) with small autapomorphic segregates. Croton s.l. appears to have a EuphorbioideaeÐEuphorbioideae are distinguished from New World origin and to have dispersed into the Old World other subfamilies of Euphorbiaceae by a combination of latex (see embedded Cr. gratissimus with trnL-F; Cr. insularis and presence, relatively homogeneous tricolporate pollen, and Colobocarpos with rbcL). The positions of Croton lobatus highly reduced staminate ¯owers lacking enough perianth to (Croton section Astraea) and the Cuban endemic Moacroton cover the anthers. They contain a remarkable array of in¯o- indicate that further adjustments are needed to make Croton rescence modi®cations including several independent conden- monophyletic. These include subsuming Moacroton or enlarg- sations resulting in pseudanthia and other aggregated forms ing that genus to include Croton alabamensis and its allies (e.g., Maprounea and Hura) as well as pollinator specializa- identi®ed in detailed molecular phylogenetic studies, and the tions (i.e., Hura, Mabea, and Pedilanthus). The presence of generic segregation of Croton section Astraea (i.e., resurrec- often highly elaborated latex starch grains (Mahlberg, 1975) tion of Astraea; P. Berry, A. Hipp, K. Wurdack, B. Van Ee, may be synapomorphic, but their distribution outside of Eu- and R. Riina, unpublished manuscript). The latter is a distinc- phorbia s.l. has not been systematically investigated other than tive group with deeply lobed leaves, an unusual chromosome being recorded for some Hippomaneae (Metcalfe and Chalk, number (x ϭ 9; Hans, 1973), and noted as lacking the intra- 1950) and Anthostema (Mahlberg and Ake Assi, 2002). Eu- August 2005] WURDACK ET AL.ÐEUPHORBIACEAE PHYLOGENETICS 1411 phorbioideae, although only moderately supported (BP 78) due al., 1975). Its remarkable morphological radiation, including to Stomatocalyceae, were the only monophyletic subfamily of diverse succulents, and to a lesser extent cyathial modi®ca- uniovulate Euphorbiaceae s.l. recovered in our analyses. Sto- tions, has provided impetus for the recognition of autapo- matocalyceae contains four small genera (three sampled here), morphic segregates. Our sampling includes members of the three with Old World distributions and one (Nealchornea) segregates Chamaesyce, Cubanthus (rbcL only), Monadenium, from the Neotropics. The tribe has been considered discordant Pedilanthus, and Synadenium, as well as representatives of the in Euphorbioideae and even suggested to have af®nities with four major clades of Euphorbia s.l. (A, B, C, D; Steinmann Omphalea because of ``liana habit, colored latex, and oily en- and Porter, 2002). The continued recognition of Euphorbia s.l. dosperm'' (Webster, 1994b, p. 117). This associative evidence segregates would necessitate the recognition of several addi- is overstated as only one taxon is scandent (Hamilcoa, not tional subclades at generic rank, the boundaries of which are sampled), the latex is of a different color (white or yellowish not understood (see Webster, 1967, for a review of the prob- instead of red as in Omphalea), and oily endosperm is scat- lem). tered across the family in homoplasiously large-seeded taxa. The nonpseudanthial Euphorbioideae contain two strongly The remaining Euphorbioideae are strongly supported as supported subclades (H1, H2) that are novel groupings of monophyletic and divided into supported pseudanthial and members from three tribes. Hippomaneae are paraphyletic with nonpseudanthial clades (BP 81 and BP 86, respectively). In embedded Hureae ϩ Pachystromateae. The lack of apparent addition to the eponymous in¯orescence difference, the non- morphological synapomorphies for the subclades makes as- pseudanthial clade has inclinate buds (see below) and entire signment of unsampled taxa dif®cult in the fashion noted pre- styles (except some Homalanthus), whereas the pseudanthial viously for the inaperturate crotonoid subclades. Esser et al. clade has ¯owers with erect development and mostly bi®d (1997) considered inclinate staminate ¯ower buds, providing styles. Pseudanthial taxa also possess specialized pollen ap- bracteal shielding to the otherwise poorly protected ¯owers, ertures with bandlike intine thickenings that appear to be ab- to be a synapomorphy for Hippomaneae and perhaps unique sent from the few nonpseudanthial members examined (SuaÂ- in the family. However, he also recorded this character for rez-Cervera et al., 2001). The pseudanthial clade corresponds three (Algernonia, Ophthalmoblapton, and Tetraplandra)of to tribe Euphorbieae and follows the subtribal classi®cation of the four genera of Hureae except Hura (Esser in Radcliffe- Webster (1975, 1994b) that tracks increasing ¯oral reduction Smith, 2001, pp. 352±398). The reduced congested in¯ores- and involucre modi®cations in the pseudanthia. Steinmann and cences of Hureae make it dif®cult to assess this character; Porter (2002) found an identical relationship among their basal sections of immature Tetraplandra in¯orescences show no ap- nodes using nuclear ITS and plastid ndhF for well-sampled parent inclination (K. J. Wurdack, personal observation). The trees. Subtribes Anthosteminae and Neoguillauminiinae appear erect buds of Pachystromateae have been considered part of to be transitional relics in the evolution of cyathia in Euphor- the justi®cation for maintaining that monotypic tribe. Generic biinae. Euphorbia pachysantha (see Rauh, 1996) also appears delimitation in subtribe Hippomaninae has been considered to ``primitive'' by virtue of its loose cyathial structure and well- be the most problematic in Euphorbiaceae s.s. Webster (1994b) developed bracts and bracteoles, but this taxon was shown by recognized 19 genera, but the most recent classi®cation by Haevermans (2003) to be embedded in Euphorbia s.l. The cy- Esser (Radcliffe-Smith, 2001, 352±398) includes 33 genera, athium is a highly reduced pseudanthial syn¯orescence em- mostly containing one to four species. Justi®cation for these bedded in a fused involucral cup with a central pistillate ¯ower changes was partly found in morphological phylogenetic anal- surrounded by numerous cymose clustered staminate in¯ores- yses of limited sampling (Kruijt, 1996; Esser et al., 1997). cences, the staminate ¯owers being reduced to a single anther Molecular data here provide limited resolution, and nuclear and associated bracteole. The pistillate ¯owers are solitary and ITS is perhaps a more appropriate marker (see outgroups in terminal in the pseudanthial taxa but usually several and lateral Steinmann and Porter, 2002). in the thyrses of the nonpseudanthial clade. In Anthostema the Sebastiania s.l. and Sapium s.l. have been the most dis- pistillate ¯ower appears lateral, but this has not been examined sected in post-Webster classi®cations. Ten sampled species be- anatomically (see Venkata Rao, 1971); its sister in our trees, long to Sebastiania s.l. (i.e., as circumscribed by Webster, Dichostemma, has a clearly central pistillate ¯ower. The origin 1994b), including the type Sebastiania brasiliensis (trnL-F of the cyathium has been the topic of considerable debate (see only). Sebastiania bilocularis, S. cornuta, and S. lottiae (latter Gilbert, 1994), with particular concern paid to perceived in- two trnL-F only) from arid regions of southwestern North surmountable disparities of in¯orescence architecture with America group with the predominantly West Indian genera non-pseudanthial Euphorbioideae. The divergence of in¯ores- Bonania, Grimmeodendron, and Hippomane. Sebastiania bil- cence types appears old with macrofossils evident in Middle ocularis was tentatively referred to the newly described Pler- Eocene ¯oras of Tennessee (nonpseudanthial Hippomaneo- adenophora, but the nomenclatural combination was not for- idea, Crepet and Daghlian, 1982; pseudanthia, Friis and Cre- mally made (Esser in Radcliffe-Smith, 2001, pp. 352±398, as pet, 1987). The in¯orescences of Stomatocalyceae are unisex- Sapium biloculare); Sebastiania lottiae has also been suggest- ual and do not shed any light on the ancestral in¯orescence ed to belong there (V. Steinmann, Instituto de EcologõÂa, A.C., type that became condensed in Euphorbieae. Our data con®rm Mexico, personal communication) based on its morphological that Euphorbia s.s. is paraphyletic and that all sampled cy- characters. A strongly supported grouping unites the segre- athial taxa form a strongly supported monophyletic group (i.e., gates Microstachys and Ditrysinia with aberrant Sebastiania Euphorbia s.l.). Steinmann and Porter (2002) reached the same hexaptera (trnL-F only; see Esser, 1999). Microstachys is the conclusion from more broadly sampled analyses. Euphorbia most distinctive Sebastiania segregate and has the greatest di- s.l. is among the largest genera of ¯owering plants with 1836 versity in Brazil (Esser, 1998). Caribbean Sebastiania hexap- species currently recognized (Govaerts et al., 2000) and nearly tera is morphologically and biogeographically closer to mono- 10 000 names (Oudejans, 1990). It is also the only genus with typic Ditrysinia from the southeastern United States. The two all three photosynthetic systems (CAM, C3, C4; Webster et remaining species of Sebastiania included in our analyses are 1412 AMERICAN JOURNAL OF BOTANY [Vol. 92 found in groupings with Gymnanthes glabrata and Mabea on taining the majority of the species, may deserve to be rein- the one hand and with Excoecaria and Spirostachys on the stated at the generic level (see Jablonski, 1969) and is distin- other hand. The current concept of Sebastiania clearly needs guished by brownish indument, appendiculate ovary/fruit and to be further examined. The strongly supported position of a horizontal embryo (the last character is poorly known) vs. Mabea among nondescript Sebastiania segregates does not il- Actinostemon lacking indument, with smooth ovary/fruit and luminate the orgins of its ¯oral specializations for animal pol- a vertical embryo. lination (see Vieira and de Carvalho-Okano, 1996). Hureae are paraphyletic due to embedded Pachystromateae Sapium s.l. (sensu Webster) has been split into eight genera, and are in turn part of hippomanoid subclade H2. Both tribes and this appears to be at least partly supported by our results share a Neotropical distribution, large woody fruits, and ¯ow- in which the four segregates sampled (Neoshirakia, Sapium ers with varying degrees of style, calyx, and stamen fusion. s.s., Sclerocroton, and Triadica) fall in three places. Sapium Pachystroma is a spiny-leaved shrub or small tree from south- s.s. (sensu Kruijt, 1996) is embedded in Stillingia as may be eastern Brazil and adjacent Bolivia, and also occurs disjunct Falconeria (not sampled; Esser et al., 1997; Esser, 1999) that in Peru. Webster (1994b) considered the monotypic Pachys- has intermediate features. Sapium s.s. possesses red-arillate tromateae to be a doubtfully distinct tribe, but Esser (Rad- seeds, no carpidiophore, and pollen with an equatorial ring, cliffe-Smith, 2001, 352±398) reaf®rmed its distinctiveness whereas Stillingia has nonarillate (carunculate or ecarunculate) from Hippomaneae based on ¯oral differences. Molecular data seeds, a carpidiophore, and pollen lacking an equatorial ring, strongly support the sister status of Pachystroma with Tetra- although the last character is poorly known and needs to be plandra (and presumably closely related if not congeneric Al- compared with Euphorbieae intine thickenings (see SuaÂrez- gernonia, not sampled) ϩ Ophthalmoblapton in Hureae. This Cervera et al., 2001). group also shares fruits with carpidiophores, although clearly Stillingia is easily distinguished by a carpidiophore (gyno- different from those noted previously in Stillingia and Aden- base, sensu Rogers, 1951), a persistent, woody, triangular fruit opeltis. Hura, the well-known sand-box tree, is the most spe- base. Stillingia and Adenopeltis both have a carpidiophore, but cialized taxon in Hippomaneae with massive aggregated in¯o- historically they have been considered distinct genera mainly rescences that are bat-pollinated (Steiner, 1982). Webster's due to staminate perianth differences (i.e., naked ¯owers in suggestion (1994b) that Hureae closely approach Hippoma- Adenopeltis and calycine in Stillingia). Our analyses show that neae through Dalembertia is not supported; Dalembertia be- Stillingia is paraphyletic in a strongly supported nearly exclu- longs in the other hippomanoid subclade (H1). sively Neotropical clade with Adenopeltis, Spegazziniophytum, and Sapium s.s. All four genera share staminate ¯owers with Conclusions: towards a new phylogenetic classi®cationÐ only two stamens and fused calyces (lacking in Adenopeltis) The results presented here indicate that the current classi®ca- and (sub-) sessile pistillate ¯owers, although individually these tion of Euphorbiaceae s.s. needs revision. Broad changes characters are not unique in Hippomaneae. Stillingia includes would include the recognition of ®ve to nine subfamilies cor- three Paci®c species, with one (St. lineata, trnL-F only) sam- responding to some (preferable) or all (unwieldy) of the major pled here as the poorly supported sister to Sapium s.s. Esser clades enumerated here and a reduction in the number of (Radcliffe-Smith, 2001, 352±398) segregated Stillingia pata- tribes. We consider it premature to present a new complete gonica as monotypic Spegazziniophytum for appearing to lack classi®cation at this time due to lack of supported relationships a carpidiophore. This highly aberrant Patagonian shrub has for Erismantheae and among the Crotonoideae s.l. lineages succulent thorn-tipped photosynthetic branches with well-de- that relate to subfamily delimitation, and also poor support veloped stomata and bearing reduced leaves when young, and within core acalyphoids and inaperturate crotonoids that relate a reduced carpidiophore (K. J. Wurdack, personal observation; to tribal circumscriptions. We do, however, propose the rec- Correa, 1988, p. 84, ®g. 61g). Rogers (1951) considered it to ognition of two additional subfamilies to accommodate mem- be a Sapium, and the original description was in Colliguaja. bers of the acalyphoid grade with supported positions. Molecular data place Spegazziniophytum as the strongly sup- Subfamily Peroideae Baill. ex Hassk., Flora 42: 649 (7 Nov. ported sister to Stillingia paucidentata from California, the 1859) (as Pereae). only sampled member of subgenus Gymnostillingia. PerideÂes Baill., Etude Euphorb.: 433 (1858), nom. inval. (non- Gymnanthes and Actinostemon appear polyphyletic in our Latin ending). analyses. Gymnanthes glabrata is clearly separated from the Peraceae (Baill.) Klotzsch, Monatsber. KoÈnigl. Preuss. Akad. other sampled species and belongs to the segregate Sarothros- Wiss. Berlin: 241, 246 (10±30 March 1859). tachys. Esser (1999) united Sarothrostachys with Gymnanthes, Type: Pera Mutis. although previously it was generally accepted as an aberrant Prosopidoclineae Klotzsch, Arch. Naturgesch. 7: 176 (1841). section of Sebastiania. Gymnanthes cf. albicans (rbcL only) Type: Pera Mutis. groups with Actinostemon caribaeus and suggests the need for Genera: Chaetocarpus Thwaites, Clutia L., Pera Mutis, Po- generic evaluation of a group of Caribbean endemics that have gonophora Miers ex Benth., Trigonopleura Hook. f. been variously shifted between those genera. Actinostemon amazonicus and A. caribaeus each represent the two subgenera Dioecious (more rarely monoecious) trees, shrubs, or herbs. recognized by Pax and Hoffmann (1912): Dactylostemon (sec- Latex absent. Indumentum simple, T-shaped, stellate, or lepi- tion Armati) and Euactinostemon (section Inermes), respec- dote. Leaves stipulate or exstipulate, petiolate to subsessile, tively. These subgenera are morphologically distinct (evident alternate (rarely opposite in Pera), simple, unlobed, penni- in the contrasts present in Esser's generic description in Rad- nerved, eglandular, sometimes pellucid-punctate, entire. In¯o- cliffe-Smith, 2001, pp. 352±398) despite Jablonski (1969) dis- rescences axillary, without or rarely with elongated axes, sur- regarding the infrageneric groups in his revision and merely rounded by involucral bracts in Pera only. Bracts eglandular. emphasizing the sterile deciduous scales covering the in¯ores- Flowers: sepals 2±6 (absent in pistillate ¯owers and sometimes cence buds as a good generic character. Dactylostemon, con- rudimentary in staminate ¯owers of Pera), imbricate; petals August 2005] WURDACK ET AL.ÐEUPHORBIACEAE PHYLOGENETICS 1413 present or absent; disc present (absent in Pera); stamens 2± ular phylogenetic studies will need to be well-sampled and not 20, free to connate; anthers introrse or extrorse; pollen prolate rely on generic exemplars as many often poorly known taxa to oblate spheroidal, 3-(rarely 4-)colporate, mostly tectate-per- have yet to placed within the framework of the current Hip- forate, sometimes almost tectate or ®nely reticulate, mostly pomaneae classi®cation. All generic segregates of Euphorbia psilate (microrugulate in Chaetocarpus), intectate in Pera ar- would have to be subsumed in order to make that genus mono- borea; pistillode present (absent in Pera, but reduced pistillate phyletic, as has been recently recommended by Steinmann ¯owers surrounding staminate ¯owers in some species); ovary (Steinmann and Porter, 2002; Steinmann, 2003). Tribes that 3(±4)-locular, styles bi®d to bipartite (short with umbraculi- are clearly not monophyletic include Acalypheae, Adenocli- form stigma in Pera); ovules 1 per locule. Fruit dehiscent to neae, Agrostistachydeae, Aleuritideae, Bernardieae, Caryoden- sometimes incompletely dehiscent; septa membranous, fragile, dreae, Chrozophoreae, Codiaeae, Crotoneae, Hippomaneae, without visible vascularisation; valves often remaining at- Hureae, Jatropheae, Micrandreae, and Pycnocomeae. Subsum- tached to the base of the columella after dehiscence. Seeds ing the monogeneric tribes Omphaleae and Pachystromateae shiny, black, smooth, carunculate; endosperm copious in ma- and modest generic realignments are supported by our data. ture seed (but scanty in Trigonopleura); cotyledons longer and Relationships are poorly resolved within the core acalyphoid wider than radicle. and inaperturate crotonoid clades, but groupings corresponding to current tribes or subtribes are apparent. Para- and/or poly- Subfamily Cheilosoideae (MuÈll. Arg.) K. Wurdack & Petra phyletic genera include Actinostemon, Alchornea, Croton, Eu- Hoffm., stat. nov. phorbia, Excoecaria, Gymnanthes, Sebastiania, Stillingia, Te- Hippomaneae subtribe Cheiloseae MuÈll. Arg., Linnaea 34: 202 trorchidium, and Tragia. Differences in generic circumscrip- (1865). Gelonieae subtribe Chaetocarpinae ser. Cheilosifor- tions between Webster (1994b) and Radcliffe-Smith (2001) mes (MuÈll. Arg.) Pax & K. Hoffm., P¯anzenr. 68, Addit. need to be evaluated in a better-sampled phylogenetic context, 6: 50 (1919). Cheiloseae (MuÈll. Arg.) Airy Shaw & G. L. but many of the generic segregates resurrected by the latter Webster, Taxon 24: 595 (1975). appear unwarranted. Type: Cheilosa Blume. Genera: Cheilosa Blume, Neoscortechinia Hook. f. ex Pax. LITERATURE CITED Dioecious trees. Latex absent. Indumentum simple and/or stellate. Leaves stipulate, petiolate, alternate, simple, unlobed, ADAMS, K. L., Y.-L. QIU,M.STOUTEMYER, AND J. D. PALMER. 2002. Punc- penninerved, usually glandular at the base of the lamina, entire tuated evolution of mitochondrial gene content: high and variable rates or toothed with glands situated on the abaxial side of teeth. of mitochondrial gene loss and transfer to the nucleus during angiosperm In¯orescences pseudoterminal or axillary, with or without evolution. Proceedings of the National Academy of Sciences, USA 99: 9905±9912. elongated axes. Bracts eglandular. Flowers: sepals 4±6, im- ANGIOSPERM PHYLOGENY GROUP (APG II). 2003. An update of the Angio- bricate; petals absent; disc present in staminate ¯owers, pre- sperm Phylogeny Group classi®cation for the orders and families of ¯ow- sent or absent in pistillate ¯owers; stamens 5±12, free; anthers ering plants: APG II. Botanical Journal of the Linnean Society 141: 399± introrse; pollen 2-nucleate, globose, tricolporate, echinate; pis- 436. tillode present; ovary 2±4-locular, styles bi®d; ovules 1 per ARMBRUSTER, W. S. 1996. Cladistic analysis and revision of Dalechampia locule, anatropous, vascular bundles present in outer integu- sections Rhopalostylis and Brevicolumnae (Euphorbiaceae). Systematic Botany 21: 209±235. ment. Fruit tardily loculicidally dehiscent. Seeds ecarunculate, ARMBRUSTER, W. S. 2002. Can indirect selection and genetic context con- with sarcotesta; endosperm present in mature seed; cotyledons tribute to trait diversi®cation? A transition-probability study of blossom- longer and wider than radicle. colour evolution in two genera. Journal of Evolutionary Biology 15: 468± Regrettably, no DNA of the rare, monotypic genus Cheilosa 486. could be obtained for our molecular analyses. We have, how- ARMBRUSTER,W.S.,AND B. G. BALDWIN. 1998. Switch from specialized to ever, decided to raise Cheiloseae to subfamilial rank rather generalized pollination. Nature 394: 632. BALAJI, K., R. B. SUBRAMANIAN, AND J. A. INAMDAR. 1996. Occurrence of than coin the unwieldy ``Neoscortechinioideae,'' mainly on the laticifers in Kirganelia reticulata (Poir.) Baill. (Euphorbiaceae). Phyto- grounds that both genera share the unique pollen type and are morphology 46: 81±84. palynologically almost indistinguishable (Takahashi et al., BENTHAM, G. 1878. Notes on Euphorbiaceae. Journal of the Linnean Society, 1995; Nowicke et al., 1998). 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VAN, AND W. S TUPPY. 1999. Phylogenetic considerations of KOWKI. 2000. Pollen morphology, exine structure, and systematics of Euphorbiaceae tribe Aleuritideae. Annals of the Missouri Botanical Gar- Acalyphoideae (Euphorbiaceae), part 3: Tribes Epiprineae (Epiprinus, den 86: 894±903. Symphyllia, Adenochlaena, Cleidiocarpon, Koilodepas, Cladogynos, Ce- WIKSTROÈ M, N., V. SAVOLAINEN, AND M. W. CHASE. 2001. Evolution of the phalocrotonopsis, Crotonogynopsis, Enriquebeltrania, Lasiocroton, Leu- angiosperms: calibrating the family tree. Proceedings of the Royal So- cocroton), Alchorneae (Or®lea, Alchornea, Coelebogyne, Aparisthmium, ciety of London, Series B 268: 2211±2220. Bocquillonia, Conceveiba, Gavarretia), Acalypheae pro parte (Ricinus, WURDACK, K. J. 2002. Molecular systematics and evolution of Euphorbi- Adriana, Mercurialis, Leidesia, Seidelia, Dysopsis, Wetria, Cleidion, aceae sensu lato. Ph.D. dissertation, University of North Carolina, Chapel Sampantaea, Macaranga). Review of Palaeobotany and Palynology 110: Hill, North Carolina, USA. 1±66. WURDACK,K.J.,AND M. W. CHASE. 1996. Molecular systematics of Eu- THOMPSON, J. D., T. J. GIBSON,F.PLEWNIAK,F.JEANMOUGIN, AND D. G. phorbiaceae sensu lato using rbcL sequence data. American Journal of HIGGINS. 1997. The Clustal X windows interface: ¯exible strategies for Botany 83 (Supplement). 203 (Abstract). multiple sequence alignment aided by quality analysis tools. Nucleic Ac- WURDACK,K.J.,AND M. W. CHASE. 1999. Spurges split: molecular system- ids Research 25: 4876±4882. atics and changing concepts of Euphorbiaceae, s.l. XVI International Bo- tanical Congress, St. Louis. Missouri, USA. Abstracts, 142. THURSTON, E. L. 1976. Morphology, ®ne structure and ontogeny of the sting- ing emergence of Tragia ramosa and T. saxicola (Euphorbiaceae). Amer- WURDACK,K.J.,AND M. W. CHASE. 2002. Phylogenetics of Euphorbiaceae s.s. using plastid (rbcL and trnL-F) DNA sequences. Botany 2002, annual ican Journal of Botany 63: 710±718. meeting of Botanical Society of America, Madison, Wisconsin, USA. TOKUOKA,T.,AND H. TOBE. 1998. Ovules and seeds in Crotonoideae (Eu- Abstracts, 160. Avilable at website, http://www.botany2002.org/. phorbiaceae): structure and systematic implications. Botanische Jahr- WURDACK, K. J., P. HOFFMANN,R.SAMUEL,A.DE BRUIJN,M.VAN DER buÈcher fuÈr Systematik, P¯anzengeschichte und P¯anzengeographie 120: BANK, AND M. W. CHASE. 2004. Molecular phylogenetic analysis of 165±196. Phyllanthaceae (Phyllanthoideae pro parte, Euphorbiaceae sensu lato) us- TOKUOKA,T.,AND H. TOBE. 1999. Embryology of tribe Drypeteae, an enig- ing plastid rbcL DNA sequences. American Journal of Botany 91: 1882± matic taxon of Euphorbiaceae. Plant Systematics and Evolution 215: 1900. 189±208. ZUKER, M. 2003. Mfold web server for nucleic acid folding and hybridization TOKUOKA,T.,AND H. TOBE. 2001. Ovules and seeds in subfamily Phyllan- prediction. Nucleic Acids Research 31: 3406±3415. thoideae (Euphorbiaceae): structure and systematic implications. Journal of Plant Research 114: 75±92. TOKUOKA,T.,AND H. TOBE. 2002. Ovules and seeds in Euphorbioideae (Eu- phorbiaceae): structure and systematic implications. Journal of Plant Re- APPENDIX. Taxa analyzed in this study including voucher information and search 115: 361±374. GenBank sequence accession numbers. Generic circumscriptions follow TOKUOKA,T.,AND H. TOBE. 2003. Ovules and seeds in Acalyphoideae (Eu- Radcliffe-Smith (2001); Govaerts et al. (2000) was used for speci®c no- phorbiaceae): structure and systematic implications. Journal of Plant Re- menclature. Nomenclatural differences between these authorities are not- search 116: 355±380. ed as are name changes for previously published data. (a) indicates DNA URBATSCH, L. E., J. D. BACON,R.L.HARTMAN,M.C.JOHNSTON,T.J. extractions from the cited herbarium specimen. All other extractions for WATSON JR., AND G. L. WEBSTER. 1975. Chromosome numbers for new data are from silica gel dried or fresh collections. Source information North American Euphorbiaceae. American Journal of Botany 62: 494± is incomplete for some published sequences. 500. VEGA, A. S., M. A. CASTRO, AND W. R. ANDERSON. 2002. Occurrence and Taxon; Origin; Source/Voucher; GenBank accession numbers: rbcL; trnL-F. phylogenetic signi®cance of latex in the Malpighiaceae. American Jour- Euphorbiaceae s.s. nal of Botany 89: 1725±1729. Acalypha californica Benth.; USA, California; G. Levin 2192 (SD); VENKATA RAO, C. 1971. Anatomy of the in¯orescence of some Euphorbi- AY794943; AY794776. Acalypha platyphylla MuÈll. Arg.; Ecuador; G. aceae with a discussion on the phylogeny and evolution of the in¯ores- Webster & Castro 32371 (DAV); AY794942; AY794775. Acalypha vir- cence including the cyathium. Botaniska Notiser 124: 39±64. ginica L. var. rhomboidea (Raf.) Cooperr. [as Acalypha rhomboidea Raf.]; VIEIRA,M.F.,AND R. M. DE CARVALHO-OKANO. 1996. Pollination biology Unknown; voucher not seen.; L. Gunter 495 (UGA); U00435, Gunter et of Mabea ®stulifera (Euphorbiaceae) in southeastern Brazil. Biotropica al., 1994;Ð. Actinostemon amazonicus Pax & K. Hoffm.; Venezuela, 28: 61±68. Amazonas; G. Zimmermann et al. 52/152/89 (USa); AY794861; AY794655. WEBSTER, G. L. 1967. The genera of Euphorbiaceae in the southeastern Unit- Actinostemon caribaeus Griseb.; Panama; C. Galdames 3568 (NYa); ed States. Journal of the Arnold Arboretum 48: 303±430. AY794863; AY794657. Adelia ricinella L.; Puerto Rico; F. Axelrod 3874 WEBSTER, G. L. 1975. Conspectus of a new classi®cation of the Euphorbi- (NYa); AY794918; AY794737. Adenochlaena leucocephala Baill.; Ma- aceae. Taxon 24: 593±601. yotte; F. Barthelat 758 (Ka); AY794970;Ð. Adenocline pauci¯ora Turcz.; WEBSTER, G. L. 1987. The saga of the spurges: a review of classi®cation South Africa, Cape Province; J. Vlok 2147 (MOa);Ð; AY794670. Aden- and relationships in the Euphorbiales. Botanical Journal of the Linnean ocline violifolia (Kunze) Prain; South Africa, Western Cape; P. Goldblatt Society 94: 3±46. & J. Manning 10677 (MOa); AY794870; AY794669. Adenopeltis serrata WEBSTER, G. L. 1993. A provisional synopsis of the sections of the genus (Aiton) I. M. Johnst.; Chile; L. Landrum 5925 (RSAa); AY794844; Croton (Euphorbiaceae). Taxon 42: 793±823. AY794633. Adenophaedra grandifolia (Klotzsch) MuÈll. Arg.; Costa Rica; WEBSTER, G. L. 1994a. Classi®cation of the Euphorbiaceae. Annals of the O. Valverde 48 (NYa); AY794930; AY794758. Adriana tomentosa Missouri Botanical Garden 81: 3±32. (Thunb.) Gaudich. var. tomentosa ; Cult. Australia, Austr. Nat. Botanical WEBSTER, G. L. 1994b. Synopsis of the genera and suprageneric taxa of Gardens, Canberra; K. Cameron s.n. (US); AY794917; AY794736. Al- Euphorbiaceae. Annals of the Missouri Botanical Garden 81: 33±144. chornea cordifolia (Schumach. & Thonn. in C.F. Schumacher) MuÈll. Arg.; WEBSTER, G. L., W. V. BROWN, AND B. N. SMITH. 1975. Systematics of Gabon; G. Walters et al. 1000 (MO);Ð; AY794797. Alchornea laxi¯ora photosynthetic carbon ®xation pathways in Euphorbia. Taxon 24: 27±33. (Benth.) Pax & K. Hoffm.; Zaire; R. Gereau et al. 5346 (MOa); AY794957; WEBSTER, G. L., M. J. DEL-ARCO-AGUILAR, AND B. A. SMITH. 1996. Sys- AY794795. Alchornea sp.; Peru; D. Bell et al. 94±103 (US); AY794956; tematic distribution of foliar trichome types in Croton (Euphorbiaceae). Ð. Alchorneopsis ¯oribunda (Benth.) MuÈll. Arg.; French Guiana; S. Mori Botanical Journal of the Linnean Society 121: 41±57. et al. 21587 (NYa); AY794962; AY794800. Aleurites moluccana (L.) 1418 AMERICAN JOURNAL OF BOTANY [Vol. 92

Willd.; Cult. USA, Missouri, MO 895208; K. Wurdack (US); AY794883; AY794692. Croton cuneatus Klotzsch; Venezuela, Apure; P. Berry 7589 AY794709. Amperea xiphoclada (Sieber ex Spreng.) Druce; Cult. Indo- (PORT);Ð; AY794698. Croton gratissimus Burch.; Cult. USA, New York, nesia, Bogor Botanic Garden IX.A.64a; Chase 1936 (K); AY794911;Ð. NYBG; K. Wurdack D536 (US);Ð; AY794696. Croton heliotropiifolius Amyrea sambiranensis Leandri; Comoro Islands, Mayotte; O. Pascal 659 Kunth; Venezuela; P. Berry 5542 (MO);Ð; AY794695. Croton lobatus L.; (NYa); AY794964; AY794801. Anthostema senegalense A. Juss.;Ð; [S. Venezuela; R. Riina 1268 (VEN, WIS);Ð; AY794689. Croton lobatus L.; Renner]; AY794830; AY794609. Aparisthmium cordatum (A. Juss.) Baill.; US Virgin Islands, St. Croix; V. Steinmann 2024 (RSAa); AY794905;Ð. Peru; D. Bell et al. 93-60 (US); AY794955; AY794793. Aparisthmium sp. Croton lucidus L.; Cult. USA, Florida, Fairchild Tropical Garden 76417a; nov. ined.; Brazil; A. Carvalho et al. 4503 (NYa); AY794954; AY794792. K. Wurdack D117 (US); AY794909;Ð. Croton lucidus L.; Puerto Rico, Argomuellera sp.; Madagascar; G. McPherson 18425 (MO); AY794938; Cabo Rojo; M. Nee 44194 (MO);Ð; AY794701. Croton cf. olivaceus MuÈll. Ð. Argomuellera macrophylla Pax; Ghana; H. Schmidt et al. 2240 (MO); Arg.; Ecuador, Napo; D. Neill 11163 (MOa);Ð; AY794694. Croton spe- AY794937; AY794769. Astrococcus cornutus Benth.; Venezuela, Ama- ciosus MuÈll. Arg.; Venezuela, Districto Federal; P. Berry 7590 (MO);Ð; zonas; R. Leisner 8693 (NYa); AY794928; AY794755. Aubletiana ma- AY794699. Crotonogyne sp.; Gabon; S. Smith 1742 (US); AY794893; crostachys (Breteler) J. Murillo; Gabon; L. White 1190 (MOa); AY794960; AY794717. Crotonogynopsis usambarica Pax; Tanzania; R. Polhill et al. AY794798. 5129 (Ka); AY794972;Ð. Crotonopsis linearis Michx. [ϭ Croton mi- Baliospermum montanum (Willd.) MuÈll. Arg.; Laos; J. Munzinger 169 chauxii G.L. Webster]; USA, North Carolina; K. Wurdack (US);Ð; (MOa); AY794884; AY794726. Baloghia inophylla (G. Forst.) P. S. Green; AY794702. Cubanthus umbelliformis Urb. & Ekman; Cuba; HAJB 81901 Cult. Australia, Aust. Nat. Botanical Gardens, Melbourne; M. Chase 3062 (HAJB); AY794973;Ð. Cyrtogonone argentea (Pax) Prain; Nigeria; B. (K); AY794880; AY794707. Bernardia myricifolia (Scheele) S. Watson Daramola 19 (Ka); AY794974;Ð. Cyttaranthus congolensis J. LeÂonard; [as Bernardia incana K.J. Morton]; USA, Arizona; McGill & Sundell 5459 Gabon; F. Breteler 6339 (MOa); AY794965; AY794803. Dalechampia spa- (K); AJ402928, Savolainen et al., 2000;Ð. Bernardia scabra MuÈll. Arg.; thulata (Scheidw.) Baill.; Cult. USA, North Carolina; K. Wurdack D010 Brazil, Bahia; W. Thomas et al. 12615 (NYa);Ð; AY794759. Bertya ros- (US); AY788172, Davis et al., in press; AY794754. Dalembertia populi- marinifolia Planch.; Cult. Australia, Aust. Nat. Botanical Gardens, Can- folia Baill.; Mexico; J. Rzedowski 35734 (NYa); AY794857; AY794651. berra; M. Chase 1938 (K); AY794878; AY794705. Beyeria leschenaultii Dichostemma glaucescens Pierre; Gabon; J.M. & B. Reitsma 1285 (NYa); (DC.) Baill.; Cult. Australia, Aust. Nat. Botanical Gardens, Canberra; M. AY794831; AY794610. Discoclaoxylon hexandrum (MuÈll. Arg.) Pax & Chase 1939 (K); AY794879; AY794706. Blachia sinensis Gagnep.; Thai- K. Hoffm.; Ghana; H. Schmidt et al. 1666 (MO); AY794945; AY794778. land; Newman et al. 1137 (L); AY794888; AY794727. Blumeodendron Discocleidion rufescens (Franch.) Pax & K. Hoffm.; China; D. Boufford sp.; Cult. Indonesia, Bogor Botanic Garden IX.C.144; M. Chase 1252 (K); et al. 26455 (NYa); AY794952; AY794788. Discoglypremna caloneura AJ418805, Chase et al., 2002; AY794767. Bonania cubana A. Rich.; Ba- (Pax) Prain; Ghana; D. Harder et al. 2954 (MOa); AY794963; AY794802. hamas, Andros; S. Hill 3156 (NYa); AY794833; AY794613. Bocquillonia Ditaxis argothamnoides (Bertol. ex Spreng.) Radcl.-Sm. & Govaerts; USA, goniorrhachis Airy Shaw; New Caledonia; G. McPherson 18680 (MO); Florida; K. Wurdack D105 (US); AY794921; AY794741. Ditaxis simoni- AY794958; AY794796. Brasiliocroton mamoninha P.E. Berry & I. Cor- ana Casar.; Brazil, Minas Gerais; J. Pirani et al. 3729 (NYa);Ð; deiro, ined.; Brazil, MaranhaÄo; M. Lobo et al. 340 (NYa); AY794907; AY794742. Ditrysinia fruticosa (Bartram) Govaerts & Frodin [ϭ Ditrysi- AY794691. Calycopeplus casuarinoides L.S. Sm.; Cult. USA, California, nia ligustrina (Michx.) Raf.]; USA, Georgia; K. Wurdack D149 (US); RSA; V. Steinmann 1407 (RSA); AY794829; AY794608. Caperonia pal- AY794854; AY794647. Ditta myricoides Griseb.; Puerto Rico; J. CedenÄo ustris (L.) A. St.-Hil.; USA, Florida; K. Wurdack D073 (US); AY794923; s.n. (US); AY794871; AY794675. Dodecastigma amazonicum Ducke; AY794745. Caryodendron orinocense H. Karst.; Ecuador, Napo; P. Ac- French Guiana; S. Mori 22811 (USa); AY794885; AY794711. Domohinea evedo-Rdgz. & J. CedenÄo 7644 (NYa); AY794931; AY794760. Cavacoa perrieri Leandri; Madagascar; 1293 [KW403];Ð; AY794720. Dysopsis aurea (Cavaco) J. LeÂonard; Kenya; W. Luke & S. Robertson 1922 (USa); hirsuta (MuÈll. Arg.) Skottsb. [as Dysopsis glechomoides MuÈll. Arg.]; AY794889; AY794718. Cephalocroton mollis Klotzsch; Botswana; I. Chile; O. Solbrig et al. 3815 (NYa); AJ402946, Savolainen et al., 2000;Ð Grignon 205 (MOa); AY794950; AY794786. Cephalomappa malloticarpa . Dysopsis paucidentata (MuÈll. Arg.) Lozano & J. Murillo; Ecuador; A. J.J. Sm.; Cult. Indonesia, Bogor Botanic Garden VIII.F.32; M. Chase 1256 Fierro 1218 (NYa);Ð; AY794746. Elateriospermum tapos Blume; Malay- (K); AJ418807, Chase et al., 2002; AY794784. Chaetocarpus africanus sia; E. Soepadmo & S. Suhaimi s193 (NYa); AY794873; AY794678. En- Pax; Gabon; L. White 1319 (MOa); AY794969; AY794809. Chamaesyce dospermum moluccanum (Teijsm. & Binn.) Kurz; Cult. Indonesia, Bogor mesembryanthemifolia (Jacq.) Dugand [ϭ Euphorbia mesembryanthem- Botanic Garden XII.B.IX.122; M. Chase 1258 (K); AJ402950, Savolainen ifolia Jacq.]; USA, Florida; K. Wurdack D102 (US); AY794820; et al., 2000; AY794671. Enriquebeltrania crenatifolia (Miranda) Rzed.; AY794601. Chiropetalum schiedeanum (MuÈll. Arg.) Pax; Mexico; Car- Mexico, Yucatan; E. Cabrera 10768 (NYa); AY794975;Ð. Eremocarpus ranza 3565 (IEB);Ð; AY794744. Chiropetalum tricoccum (Vell.) Chodat setigerus (Hook.) Benth. [ϭ Croton setigerus Hook.]; USA, California; J. & Hassl.; Brazil, ParanaÂ; V.A. de O. Dittrich 193 (NYa); AY7947922; Hughey s.n. (US); AY794910; AY794697. Erythrococca sp.; Cameroon; AY794743. Chrozophora tinctoria (L.) Raf.; Greece; B. Verdcourt 4137 M. Cheek s.n. (K); AY794949; AY79483. Erythrococca berberidea Prain; (USa); AY794951; AY794787. Cladogelonium madagascariense Leandri; South Africa; P. C. Zietsman 4265 (NYa);Ð; AY79482. Euphorbia abys- Madagascar; R. Randrianaivo et al. 561 (MOa); AY794868; AY794667. sinica J.F. Gmel. [as Euphorbia obovalifolia A. Rich.]; Cult. UK, RBG Claoxylon australe Baill. ex MuÈll. Arg.; Cult. Australia, Aust. Nat. Botan- Kew (1986±780); M. Chase 1005 (K); AY794824;Ð. Euphorbia epithy- ical Gardens, Canberra; M. Chase 1937 (K); AY794946; AY794780. Clei- moides L. [as Euphorbia polychroma A. Kern.];Ð; M. Chase 102 (NCU); dion castaneifolium MuÈll. Arg.; Costa Rica; K. Thomsen 601 (NYa); AY788173, Davis et al., in press; AY794606. Euphorbia gymnonota Urb.; AY794936; AY794768. Clutia pulchella L.; South Africa, Bophuthatswa- Cult. USA, Florida Fairchild Tropical Garden 6448c; K. Wurdack (US); na; C. Peeters et al. 507 (NCUa); AY788168, Davis et al., in press;Ð. AY794823;Ð. Euphorbia ipecacuanhae L.; USA, South Carolina; K. Clutia pulchella L.;ÐM. Chase 5876 (K);Ð; AY794811. Clutia tomentosa Wurdack D147 (US); AY794818; AY794599. Euphorbia obesa Hook. f.; L.; South Africa, Cape Province; G. Germishuizen 6505 (MOa);Ð; Cult. USA, Maryland; K. Wurdack D539 (US); AY794826; AY794605. AY794810. Cnesmone javanica Blume; Bangladesh; A. Huq 10189 (USa); Euphorbia pulcherrima Willd. ex Klotzsch cv. `Brilliant Diamonds'; Cult. AY794926; AY794752. Cnidoscolus urens (L.) Arthur var. stimulosus USA, North Carolina; K. Wurdack D084 (US); AY794819; AY794600. (Michx.) Govaerts; USA, North Carolina; K. Wurdack D002 (US); Excoecaria sp.; Madagascar; S. Pell 678 (NY);Ð; AY794620. Excoecaria AY794874; AY794679. Codiaeum variegatum (L.) Blume; Cult. USA, agallocha L.; New Caledonia; T. Motley & K. Cameron 2053 (NY); North Carolina; K. Wurdack D033 (US); AY788169, Davis et al., in press; AY794839; AY794622. Excoecaria cochinchinensis Lour.; Cult. Indone- AY794729. Colliguaja integerrima Gillies & Hook.; Chile; L. Landrum sia, Bogor Botanic Garden II.Q.60; M. Chase 1260 (K); AJ418809, Chase 8234 (MOa); AY794836; AY794617. Colliguaja odorifera Molina; Chile; et al., 2002; AY794619. Excoecaria grahamii Stapf; Ghana; H. Schmidt L. Landrum 9815 (MOa); AY794837; AY794618. Colobocarpus nanus et al. 3362 (MOa);Ð; AY794623. Fontainea venosa Jessup & Guymer; (Gagnep.) Esser & Welzen ; Thailand; R. Pooma 4256 (BKF); AY794971; Australia, Queensland; G. Guymer 1861 (MOa); AY794881; AY794708. Ð. Conceveiba guianensis Aubl.; Suriname; J. Miller et al. 9373 (MO); Garcia nutans Vahl ex Rohr; Cult. USA, Fairchild Tropical Garden X629b; Ð; AY794791. Conceveiba martiana Baill.; Peru; D. Bell 93±176 (US); K. Wurdack D051 (US); AY794890; AY794714. Gavarretia terminalis AY788170, Davis et al., in press; AY794789. Conceveiba pleiostemona Baill.; Venezuela; P. Berry 7141 (MO); AY794953; AY794790. Gitara Donn. Sm.; Costa Rica; N. Zamora 1915 (MOa);Ð; AY794794. Croton venezolana Pax & K. Hoffm.; Peru; D. Bell et al. 94±312 (US); AY794924; alabamensis E.A. Sm. ex Chapm. var. alabamensis; Cult. USA, Maryland, AY794747. Givotia madagascariensis Baill.; Madagascar; P. Phillipson et ex Alabama; K. Wurdack D008 (US); AY788171, Davis et al., in press; al. 3767 (MOa); AY794891; AY794715. Glycydendron amazonicum August 2005] WURDACK ET AL.ÐEUPHORBIACEAE PHYLOGENETICS 1419

Ducke; Peru; L. Gillespie et al. 4546 (US); AY794876; AY794681. Gly- AY794603. Monotaxis grandi¯ora Endl.; Australia; J. Horn 2386 (DUKE); cydendron cf. amazonicum Ducke., anomalous population described by AY794912; AY794730. Monotaxis linifolia Brongn.; Australia; Coveny Mori et al., 2002; French Guiana; S. Mori et al. 23265 (USa);Ð; 11063 (RSA); AY794913; AY794731. Monotaxis megacarpa F. Muell., AY794682. Grimmeodendron eglandulosum (A. Rich.) Urb.; Cult. USA, incorrectly identi®ed as ``Poranthera sp.'' in Chase et al. (2002); Australia; Florida, Fairchild Tropical Garden 6443a; K. Wurdack D282 (US); M. Chase 2162 (K); AY794985, deposited here although ®rst published in AY794832; AY794612. Grossera macrantha Pax; Cameroon; D. Harris & Chase et al., 2002;Ð. Moultonianthus leembruggianus (Boerl. & Koord.) J. Fay 1514 (Ka); AY794976;Ð. Gymnanthes cf. albicans (Griseb.) Urb.; Steenis; Brunei; G. Challen et al. 3 (K); AY794982;Ð. Nealchornea ya- Cuba; HAJB 81718 (HAJB); AY794977;Ð. Gymnanthes glabrata (Mart.) purensis Huber; Peru; P. Fine s.n. (US); AY794865; AY794662. Neobou- Govaerts; Brazil, Bahia; A. Carvalho et al. 4606 (NYa); AY794851; tonia mannii Benth. & Hook. f. ; Central African Republic; J. Fay 6701 AY794641. Gymnanthes longipes MuÈll. Arg.; Mexico, Queretaro; B. Ser- (MOa); AY794896; AY794723. Neoguillauminia cleopatra (Baill.) Cro- võÂn 7 (MOa);Ð; AY794652. Gymnanthes lucida Sw.; Cult. USA, Florida, izat; New Caledonia; G. McPherson 17882 (MO); AY794828; AY794607. Fairchild Tropical Garden X2284a; K. Wurdack D055 (US); AY794858; Neoholstia tenuifolia (Pax) Rauschert; Malawi; E. Tawakali & I. Nacham- AY794653. Hevea brasiliensis (Willd. ex A. Juss.) MuÈll. Arg.; Unknown; ba 1706 (NYa); AY794898;Ð. Neoscortechinia kingii (Hook. f.) Pax & Unknown; Fong and Lek, 1993, in lit. (never deposited in GenBank);Ð. K. Hoffm.; Cult. Indonesia, Bogor Botanic Garden IX.B.6a; M. Chase 1265 Hevea sp. [cf. pauci¯ora (Spruce ex Benth.) MuÈll. Arg.]; Guyana; L. Gil- (K); AJ402977, Savolainen et al., 2000; AY794806. Neoshirakia japonica lespie 4272 (US); AY788175, Davis et al., in press; AY794684. Hippo- (Siebold & Zucc.) Esser; Cult. USA, North Carolina; K. Wurdack D005 mane mancinella L.; Cult. USA, Florida, Fairchild Tropical Garden (US); AY794856; AY794648. Omphalea diandra L.; Cult. UK, RBG Kew 67260a; K. Wurdack D053 (US); AY794835; AY794616. Homalanthus 1990±8160; M. Chase 570 (K); AY788183, Davis et al., in press; populneus (Geiseler) Pax; Cult. Indonesia, Bogor Botanic Garden; M. AY794672. Omphalea palmata Leandri; Madagascar; L. Gillespie 471 Chase 1266 (K); AJ402978, Savolainen et al., 2000; AY794650. Homo- (MOa);Ð; AY794673. Ophellantha steyermarkii Standl.; Mexico, Chiapas; noia riparia Lour.; Laos; Munzinger & Engelmann 162 (K); AY794978; D. Breedlove 46994 (NYa); AY794906; AY794690. Opthalmoblapton pe- Ð. Hura crepitans L.; Cult. USA, North Carolina ex Venezuela; K. Wur- dunculare MuÈll. Arg.; Brazil, Bahia; S. C. Sant'Ana 989 (NY); AY794848; dack D089 (US); AY788177, Davis et al., in press; AY794636. Hylandia AY794638. Ostodes paniculata Blume; Indonesia; M. Chase 1267 (K); dockrillii Airy Shaw; Australia, Queensland; M. Luckow 3806 (NYa); AY794900; AY794725. Pachystroma longifolium (Nees) I.M. Johnst.; AY794882; AY794710. Jatropha integerrima Jacq.; Cult. USA, Fairchild Cult. Australia; L. Prince s.n. (US); AY794847; AY794637. Paracroton Tropical Garden 63169a; K. Wurdack D047 (US); AY794902; AY794685. zeylanicus (MuÈll. Arg.) N. P. Balakr. & Chakrab.; Cult. USA, Hawaii, Joannesia princeps Vell.; Cult. Indonesia, Bogor Botanic Garden Oahu, Waimea Arb. 78S729; C. Annable 3575 (NYa); AY794894; IX.C.133; M. Chase 1262 (K), Chase et al., 2002; AJ418808, Chase et al., AY794719. Paranecepsia alchorneifolia Radcl.-Sm.; Tanzania; F. Mbago 2002; AY794686. Julocroton triqueter (Lam.) Didr. [ϭ Croton triqueter 2211 (NYa); AY794981;Ð. Pausandra martini Baill.; Venezuela, Ama- Lam.]; Bolivia; M. Nee 40034 (NYa);Ð; AY794700. Klaineanthus gabon- zonas; K. Wurdack (US); AY794887; AY794713. Pedilanthus tithymalo- iae Pierre; Gabon; L. White (ser. 2) 334 (MOa); AY794869; AY794668. ides (L.) Poit.; Cult. USA, Missouri, MO 8311489; K. Wurdack D034 (US); Koilodepas bantamense Hassk.; Cult. Indonesia, Bogor Botanic Garden AY794825; AY794604. Pera bicolor (Klotzsch) MuÈll. Arg.; Guyana; L. XII.B.VII.145; M. Chase 1263 (K); AJ402965, Savolainen et al., 2000; Gillespie 4300 (US); AY794968; AY794808. Pera sp.; Cuba; P. Delprete AY794785. Lasiocroton bahamensis Pax & K. Hoffm.; Cult. USA, Fair- et al. 8665 (NY);Ð; AY794807. Philyra brasiliensis Klotzsch; Brazil; G. child Tropical Garden 66629b; K. Wurdack D058 (US); AY788181, Davis L. Webster 25536 (NYa); AY794920; AY794740. Pimelodendron zoantho- et al., in press; AY794739. Leeuwenbergia africana Letouzy & N. HalleÂ; gyne J.J. Sm.; Cult. Indonesia, Bogor Botanic Garden XI.B.XVII.255; M. Cameroon; N. Ekema 11176 (Ka); AY794979;Ð. Leidesia procumbens (L.) Chase 1268 (K); AJ418812, Chase et al., 2002; AY794661. Plagiostyles Prain; South Africa; P. Goldblatt 7577 (MOa); AY794932; AY794761. africana (MuÈll. Arg.) Prain; Gabon; G. McPherson 16622 (MO); Leucocroton comosus Urb.; Cuba; P. Delprete et al. 8696 (NY); AY794864; AY794660. Platygyne hexandra (Jacq.) MuÈll. Arg.; Cuba; P. AY794919; AY794738. Leucocroton microphyllus (A. Rich.) Pax & K. Acevedo-Rdgz. et al. 5566 (USa);Ð; AY794751. Plukenetia volubilis L.; Hoffm.; Cuba; HAJB 81915 (HAJB); AY794980;Ð. Lobanilia bakeriana Cult. USA, Alaska; W. Armbruster 38 (?); AY794929;Ð. Pogonophora (Baill.) Radcl.-Sm.; Madagascar; J.-N. Labat & D.J. DuPuy 2354 (MOa); schomburgkiana Miers ex Benth.; French Guiana; D. Larpin 1022 (USa); AY794947; AY794779. Mabea sp.; Peru; D. Bell et al. 94±30 (US); AY788183, Davis et al., in press; AY794812. Pseudagrostistachys ugan- AY794852; AY794642. Mabea sp.; Suriname; J. Miller & W. Hauk 9335 densis (Hutch.) Pax & K. Hoffm.; Tanzania; L. Festo & C. Kayombo 441 (MO);Ð; AY794643. Macaranga grandifolia (Blanco) Merr.; Cult. USA, (MOa); AY794966; AY794804. Pseudosenefeldera inclinata (MuÈll. Arg.) Florida, Fairchild Tropical Garden 83278a; K. Wurdack D046 (US); Esser; Venezuela; K. Wurdack s.n. (US); AY794862; AY794656. Pycno- AY794935; AY794766. Mallotus japonicus (L. f.) MuÈll. Arg.; Cult. USA, coma macrophylla Benth.; Ghana; C. Jongkind et al. 1816 (MOa); North Carolina; K. Wurdack D004 (US); AY794934; AY794765. Mallotus AY794939; AY794770. Ricinocarpos tuberculatus MuÈll. Arg.; Australia; philippensis (Lam.) MuÈll. Arg.; Cult. USA, Florida Fairchild Tropical Gar- M. Chase 2164 (K); AJ418817, Chase et al, 2002.; AY794704. Ricinoden- den 96839a; K. Wurdack s.n. (US);Ð; AY794764. Manihot grahamii dron heudelotii (Baill.) Heckel; Cult. Indonesia, Bogor Botanic Garden Hook.; Cult. USA, South Carolina; K. Wurdack (US); AY794875; IX.A.64a; M. Chase 1269 (K); AY794892; AY794716. Ricinus communis AY794680. Manniophyton africanum MuÈll. Arg.; Gabon; L. White 3366 L.; Cult. USA, North Carolina; H. Hills, not vouchered; identity of source (MOa); AY794886; AY794712. Maprounea africana MuÈll. Arg.; Gabon; material visually veri®ed by K.J. Wurdack; AY788188, Davis et al., in G. Walters et al. 901 (MO);Ð; AY794659. Maprounea guianensis Aubl.; press;Ð. Ricinus communis L.; Cult. USA; K. Wurdack D009 (US); Venezuela; K. Wurdack (US);Ð; AY794658. Maprounea guianensis AY794915; AY794734. Rockinghamia brevipes Airy Shaw; Australia, Aubl.;? ; K. Kubitzki 24.11.93 (HBG); AJ418810, Chase et al., 2002;Ð. Queensland; P. Forster 27966 (NYa); AY794983;Ð. Romanoa tamnoides Mareya micrantha (Benth.) MuÈll. Arg.; Gabon; L. White 1042 (MOa); (A. Juss.) Radcl.-Sm.; Paraguay; E. Zardini & I. Chaparro 50824 (MOa); AY794941; AY794774. Mareyopsis longifolia (Pax) Pax & K. Hoffm.; Ð; AY794757. Sagotia racemosa Baill.; Peru, Madre de Dios; S. Smith Gabon; G. McPherson 16174 (MOa); AY794961; AY794799. Melanolepis 253 (USa); AY794903; AY794687. Sampantaea amenti¯ora (Airy Shaw) multiglandulosa Reichb. & Zoll.; Marianas Islands, Saipan Island; T. Mot- Airy Shaw; Thailand; C. Charoenphol et al. 4518 (NYa);Ð; AY794771. ley 2493 (NY); AY794914; AY794733. Mercurialis perennis L.; Silesia, Sandwithia guyanensis Lanj.; Guyana; R. Ek et al. 906 (NYa); AY794904; Poland; E. Koziot & K. Swierkosz 1378 (NYa); AY794944; AY794777. AY794688. Sapium glandulosum (L.) Morong; Brazil, Rio Grande do Sul; Micrandra inundata P.E. Berry & A. Wiedenhoeft; Venezuela; P. Berry R. Wasum et al. HUCS No. 11243 (USa); AY794841; AY794626. Sapium 6350 (MO); AY794877; AY794683. Micrandra siphonioides Benth. [as haematospermum MuÈll. Arg.; Bolivia; M. Nee 45872 (NYa);Ð; Micrandra minor Benth.]; Peru; Rimachi 8731 (NYa) ; AJ402974, Savo- AY794627. Sapium laurocerasus Desf.; Puerto Rico; C. Taylor 11668 lainen et al., 2000;Ð. Micrococca capensis (Baill.) Prain; Republic of (MO); AY794842; AY794628. Sclerocroton cornutus (Pax) Kruijt & Roe- South Africa, National Botanic Garden; Kurzweil 463/89 (K); AY794948; bers ; Gabon; A. Bradley et al. 1053 (MO);Ð;Ð. Sebastiania bilocularis AY794781. Microstachys corniculata (Vahl) Griseb.; Guyana; M. Jansen- S. Watson; Mexico, Sonora; V. Steinmann 1372 (RSA); AY794834; Jacobs et al. 4133 (USa); AY794855; AY794646. Milbraedia carpinifolia AY794614. Sebastiania brasiliensis Spreng.; Bolivia; M. Nee 48128 (NYa); (Pax) Hutch. var. strigosa Radcl.-Sm.; Tanzania, Pwani; P. Phillipson 4942 Ð; AY794624. Sebastiania cornuta McVaugh; Mexico, Sonora; V. Stein- (MOa);Ð; AY794722. Moacroton ekmanii (Urb.) Croizat; Cuba; F. Axel- mann 589 (RSA);Ð;Ð. Sebastiania hexaptera Urb.; West Indies, Dom- rod et al. 10371 (USa); AY794908; AY794693. Monadenium guentheri inica; C. Whitefoord 4333 (USa);Ð; AY794645. Sebastiania klotzschiana Pax; Cult. UK, RBG Kew (1957±49002); M. Chase 1007 (K); AY794822; (MuÈll. Arg.) MuÈll. Arg.; Brazil, Rio Grande do Sul; R. Wasum et al. 1966 1420 AMERICAN JOURNAL OF BOTANY [Vol. 92

(USa); AY794850; AY794640. Sebastiania lottiae McVaugh; Mexico, Mi- AY794724. Wetria australiensis P.I. Forst.; Australia; P. I. Forster 29785 choacan; V. Steinmann & E. Carranza 2353 (RSA);Ð; AY794615. Sebas- (L); AY794940; AY794772. tiania pavoniana (MuÈll. Arg.) MuÈll. Arg.; Mexico, Sinaloa; V. Steinmann 1150 (RSAa);Ð; AY794625. Seidelia triandra (E. Mey.) Pax; South West Humiriacaeae Africa; W. Giess 13381 (MOa); AY794933; AY794762. Senefelderopsis Humiria balsamifera Aubl.; Brazil; Anderson 13701 (MICH); L01926, Albert croizatii Steyerm.; Venezuela; P. Berry s.n. (MO); AY794860; AY794654. et al., 1992;Ð. Humiria balsamifera Aubl.;Ð; Anderson 13654 (MICH); Ð; AF350941, Davis et al., 2001. Humiria wurdackii Cuatrec.; Venezuela; Spathiostemon javensis Blume [as Homonoia javensis (Blume) MuÈll. K. Wurdack (US);Ð794813.P Vantanea guianensis Aubl.;Ð; Pennington Arg.]; Cult. Indonesia, Bogor Botanic Garden IX.A.26; M. Chase 1261 13855 (K); Z75679, Fay et al., 1997; AY794814. (K);Ð; AY794773. Spegazziniophytum patagonicum (Speg.) Esser; Ar- Pandaceae Galearia ®liformis (Blume) Boerl.; Cult. Indonesia, Bogor Bo- a gentina, Patagonia; R. Fortunato 4363 (US ); AY794846; AY794635. Sper- tanic Garden VII.B.44; M. Chase 1334 (K); AJ418818, Chase et al., 2002; anskia cantonensis (Hance) Pax & K. Hoffm.; China, Hunan; L. Lin-bo AY794816. Microdesmis pierlotiana J. LeÂonard; Cameroon; R. Gereau et 0139 (NYa); AY794916; AY794735. Spirostachys africana Sond.; South al. 5654 (MOa); AY663645, Wurdack et al., 2004; AY794817. Microdesmis Africa; P. Zietsman 4226 (NYa); AY794838; AY794621. Stillingia lineata puberula Hook. f. ex Planch.;Ð; M. Cheek 5986 (K); AJ402975; (Lam.) MuÈll. Arg.; Cult. Mauritius; T. Motley s.n. (US);Ð; AY794629. AJ403029, Savolainen et al., 2000. Panda oleosa Pierre; Ghana; H. Stillingia oppositifolia Baill. ex MuÈll. Arg.; Brazil, Rio Grande do Sul; A. Schmidt et al. 2048 (MOa); AY663644, Wurdack et al., 2004; AY794815. Kegler 481 (USa);Ð; AY794632. Stillingia paucidentata S. Watson; USA, California; J. Porter 11765 (RSAa); AY794845; AY794634. Stillingia syl- APPENDIX LITERATURE CITED vatica L. subsp. tenuis (Small) D. J. Rogers; USA, Florida; K. Wurdack ALBERT, V. A., S. E. WILLIAMS, AND M. W. CHASE. 1992. Carnivorous plants: D117 (US); AY794843; AY794631. Stillingia texana I. M. Johnst.; USA, phylogeny and structural evolution. Science 257: 1491±1495. Texas; T. R. van Devender 96±296 (RSAa);Ð; AY794630. Strophioblachia CHASE, M. W., S. ZMARZTY,M.D.LLEDOÂ ,K.J.WURDACK,S.M.SWENSEN, ®mbricalyx Boerl.; Cult. Indonesia, Bogor Botanic Garden VIII.F.43; M. AND M. F. FAY. 2002. When in doubt, put it in Flacourtiaceae: a molecular Chase 1270 (K); AY794901; AY794728. Sumbaviopsis albicans (Blume) phylogenetic analysis based on plastid rbcL DNA sequences. Kew Bulletin J.J. Sm.; Cult. Indonesia, Bogor Botanic Garden VIII.F.81; M. Chase 1271 57: 141±181. (K); AJ418811, Chase et al., 2002; AY794732. Suregada aequoreum DAVIS, C. C., W. R. ANDERSON, AND M. J. DONOGHUE. 2001. Phylogeny of (Hance) Seem.; Taiwan; H.-L. Ho 1004 (MOa); AY794867; AY794666. Malpighiaceae: evidence from chloroplast ndhF and trnL-F nucleotide Suregada africana (Sond.) MuÈll. Arg.; South Africa, Natal; K. & M.-J. sequences. American Journal of Botany 88: 1830±1846. Balkwill 5036 (RSAa);Ð; AY794665. Suregada boiviniana Baill.; Mada- DAVIS, C. C., C. O. WEBB,K.J.WURDACK,C.A.JARAMILLO, AND M. J. gascar; P. Rakotomalaza et al. 1292 (MOa); AY788189, Davis et al., in DONOGHUE. 2005. Explosive radiation of Malpighiales supports a mid- press; AY794663. Suregada eucleoides Radcl.-Sm.; Madagascar; D. K. Cretaceous origin of tropical rain forests. American Naturalist 165: E36± E65. Harder et al. 1569 (MOa);Ð; AY794664. Suregada glomerulata (Blume) FAY, M. F., S. M. SWENSEN, AND M. W. CHASE. 1997. Taxonomic af®nities Baill.; Cult. Indonesia, Bogor Botanic Garden VIII.F.124a; M. Chase 1272 of Medusagyne oppositifolia (Medusagynaceae). Kew Bulletin 52: 111±120. (K); AY794866;Ð. Syndyophyllum occidentale (Airy Shaw) Welzen; Sa- FONG,C.K.,AND K. C. LEK. 1993. Nucleotide sequence of the chloroplast bah; J. Aik L. Lowa & K. Kulit SAN- 111913 (L); AY794967;Ð. Syna- gene for the large sub-unit of ribulose-1,5-bisphosphate carboxylase/ denium grantii Hook. f.; Cult. USA, North Carolina, DUKE-; K. Wurdack oxygenase from Hevea brasiliensis. Journal of Natural Rubber Research (US); AY794821; AY794602. Tannodia cordifolia (Baill.) Baill.; Mayotte, 8: 146±153. a Comoro Islands; O. Pascal 656 (NY ); AY794897; AY794721. Tetraplan- GOVAERTS, R., D. G. FRODIN, AND A. RADCLIFFE-SMITH. 2000. World dra sp.; Brazil; J. G. Jardin 621 (NYa); AY794849; AY794849. Tetror- checklist and bibliography of Euphorbiaceae (with Pandaceae). 4 vols. chidium gabonense Breteler; Gabon; F. Breteler et al. 12904 (USa); Royal Botanic Gardens, Kew. AY794872; AY794674. Tetrorchidium cf. macrophyllum MuÈll. Arg.; Peru, GUNTER, L. E., G. KOCHERT, AND D. E. GIANNASI. 1994. Phylogenetic Loreto; D. Bell et al. 93±204 (US); AY788191, Davis et al., in press; relationships of the Juglandaceae. Plant Systematics and Evolution 192: 11±29. AY794676. Tetrorchidium sp. nov. ined.; Ecuador, Esmeraldas; J. Clark MORI, S. A., G. CREMERS,C.A.GRACIE, J.-J. DE GRANVILLE,S.V.HEALD, 2566 (USa);Ð; AY794677. Thyrsanthera suborbicularis Pierre ex Gag- M. HOFF, AND J. D. MITCHELL. 2002. Guide to the vascular plants of nep.; Thailand; T. Sorensen et al. 3612 (Ka); AY794984;Ð. Tragia fallax central French Guiana. Part 2. Dicotyledons. Memoirs of the New York MuÈll. Arg.; Peru, Loreto; D. Bell et al. 94±378 (US);Ð; AY794750. Tragia Botanical Garden 76: 1±776. urens L.; USA, Virginia; M. Strong 2273 (USa);Ð; AY794749. Tragia RADCLIFFE-SMITH, A. 2001. Genera Euphorbiacearum. Royal Botanic urticifolia Michx.; USA, North Carolina; K. Wurdack D074 (US); Gardens, Kew, UK. AVOLAINEN, V., M. F. FAY,D.C.ALBACH,A.BACKLUND,M.VAN DER AY794925; AY794748. Tragiella anomala (Prain) Pax & K. Hoffm.; Tan- S BANK,K.M.CAMERON,S.A.JOHNSON,M.D.LLEDOÂ , J.-C. PINTAUD,M. zania; L. Mwasumbi et al. 16202 (MOa); AY794927; AY794753. Trewia POWELL,M.C.SHEAHAN,D.E.SOLTIS,P.WESTON,W.M.WHITTEN,K. nudi¯ora L.; Cult. Indonesia, Bogor Botanic Garden III.D.7; M. Chase J. WURDACK, AND M. W. CHASE. 2000. Phylogeny of the eudicots: a nearly 1273 (K); AY663648, Wurdack et al., in press; AY794763. Triadica se- complete familial analysis based on rbcL gene sequences. Kew Bulletin 55: bifera (L.) Small; Cult. USA, North Carolina; K. Wurdack D059 (US); 257±309. AY794859; AY794649. Trigonostemon verrucosus J.J. Sm.; Cult. Indo- WURDACK, K. J., P. HOFFMANN,R.SAMUEL,A.DE BRUIJN,M.VAN DER nesia, Bogor Botanic Garden VIII.E.16; M. Chase 1274 (K); AY788192, BANK, AND M. W. CHASE. 2004. Molecular phylogenetic analysis of Davis et al., in press; AY794703. Vernicia montana Lour.; Cult. Indonesia, Phyllanthaceae (Phyllanthoideae pro parte, Euphorbiaceae sensu lato) using Bogor Botanic Garden XX.C.52a; M. Chase 2105 (K); AY794899; plastid rbcL DNA sequences. American Journal of Botany 91: 1882±1900.