PHYLOGENETICS OF SEED Mark W. Chase,2 Douglas E. Soltis,3 PLANTS: AN ANALYSIS OF Richard G. Olmstead,4 David Morgan,3 Donald H. Les,5 Brent D. Mishler, 6 NUCLEOTIDE SEQUENCES Melvin R. Duvall,7 Robert A. Price,8 FROM THE PLASTID Harold G. Hills,2 Yin-LongQiu,2 GENE rbcL' Kathleen A. Kron,2 Jeffrey H. Rettig,9 Elena Conti,10Jeffrey D. Palmer,8 James R. Manhart,9 Kenneth J. Sytsma,10 Helen J. Michaels,"1 W. John Kress,12 Kenneth G. Karol,10 W. Dennis Clark,13 Mikael Hedren,14 Brandon S. Gaut,7 Robert K. Jansen,15 Ki-Joong Kim,15 Charles F. Wimpee,5 James F. Smith,12 Glenn R. Furnier,16 Steven H. Strauss,17 Qiu-Yun Xiang,3 Gregory M. Plunkett,3 Pamela S. Soltis,3 Susan M. Swensen,18 Stephen E. Williams,19 Paul A. Gadek,20 Christopher J. Quinn,20 Luis E. Eguiarte,7 Edward Golenberg,21 Gerald H. Learn, Jr.,7 Sean W. Graham,22 Spencer C. H. Barrett,22 Selvadurai Dayanandan,23 and Victor A. Albert2

ABSTRACT

We present the results of two exploratory parsimony analyses of DNA sequences from 475 and 499 species of seed plants, respectively, representing all major taxonomic groups. The data are exclusively from the chloroplast gene rbcL, which codes for the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCOor RuBPCase). We used two different state-transformation assumptions resulting in two sets of cladograms: (i) equal-weighting for the 499-taxon analysis; and (ii) a procedure that differentially weights transversions over transitions within characters and codon positions among characters for the 475-taxon analysis. The degree of congruence between these results and other molecular, as well as morphological, cladistic studies indicates that rbcL sequence variation contains historical evidence appropriate for phylogenetic analysis at this taxonomic level of sampling. Because the topologies presented are necessarily approximate and cannot be evaluated adequately for internal support, these results should be assessed from the perspective of their predictive value and used to direct future studies, both molecular and morphological. In both analyses, the three genera of Gnetales are placed together as the sister group of the flowering plants, and the anomalous aquatic Ceratophyllum (Ceratophyllaceae) is sister to all other flowering plants. Several major lineages identified correspond well with at least some recent taxonomic schemes for angiosperms, particularly those of Dahlgren and Thorne. The basalmost clades within the angiosperms are orders of the apparently polyphyletic subclass Magnoliidae sensu Cronquist. The most conspicuous feature of the topology is that the major division is not monocot versus dicot, but rather one correlated with general pollen type: uniaperturate versus triaperturate.The Dilleniidae and Hamamelidae are the only subclasses that are grossly polyphyletic; an examination of the latter is presented as an example of the use of these broad analyses to focus more restricted studies. A broadly circumscribed Rosidae is paraphyletic to Asteridae and Dilleniidae. Subclass Caryophyllidae is monophyletic and derived from within Rosidae in the 475-taxon analysis but is sister to a group composed of broadly delineated Asteridae and Rosidae in the 499-taxon study.

I The authors acknowledge the following support: U.S. National Science Foundation (NSF) grant BSR-8906496 to MWC; BSR-9007614 to DES; BSR-9002321 to MRD; BSR-9107827 to RGO; BSR-8817992 to DHL and CFW; BSR-9107484 to BDM; BSR-9007293 and BSR-9020055 to KJS; BSR-8821264 to KAK; BSR-8717600 and BSR- 8996262 to JDP; BSR-9020171 to RKJ; BSR-8817953 to SHS and RAP and BSR-8957023 to SHS; BSR-880193 to W. Alverson; a Sloan Postdoctoral Fellowship to KJK; BSR-8914635 to VAA; a grant from the American Philosophical Society to SEW; Australian Research Council grant AD9031851 to PAG and CJQ; an operating grant to SCHB and a graduate scholarship to SWG from the Natural Sciences & Engineering Research Council of Canada; a grant to MWC and doctoral fellowship to VAA from the American Orchid Society; and a postdoctoral scholarship

ANN. MISSOURIBOT. CARD. 80: 528-580. 1993. Volume 80, Number 3 Chase et al. 529 1993 Phylogenetics of Seed Plants

Current assessments of higher-level relationships taxa from 135 families, still less than a third of in seed plants are based largely on informed judg- angiosperm families. If insufficient sampling of taxa ments of the relative value of various reproductive or characters (i.e., sequence length, acknowledged and vegetative characters (including secondary as a problem with the rbcS data studied by Martin chemistry) and to some extent on historical pre- & Dowd, 1991) are indeed factors, then no valid cedent. Authors of recent taxonomic schemes (for investigation of the "informativeness" of a given example, Dahlgren, 1980; Takhtajan, 1980, 1987; gene sequence exists for seed plants. Thus we are Cronquist, 1981; Thorne, 1983, 1992) have syn- left with only an unfounded assessment of sequence thesized an enormous amount of information. Nev- data as having the potential to aid in estimating ertheless, their taxonomic decisions have been guid- higher-level relationships. ed by estimations of which characters are reliable Suggestions that the chloroplast gene rbcL, which indicators of relationships. These differing judg- codes for the large subunit of ribulose- 1 ,5-bis- ments are responsible for radical differences in phosphate carboxylase/oxygenase (RuBisCO or delimitation and relative ranks of taxa in each RuBPCase), was an appropriate locus to use in system of classification. Recently, a number of phylogenetic studies began with Ritland & Clegg explicit, cladistic hypotheses have been developed (1987) and Zurawski & Clegg (1987). Initial at- at inclusive hierarchical levels (e.g., Crane, 1985, tempts to evaluate relationships used only a dozen 1988; Dahlgren & Bremer, 1985; Dahlgren et al., or so sequences representing all land plants (Palmer 1985; Doyle & Donoghue, 1986, 1992; Bremer et al., 1988; Giannasi et al., 1992). Other recent et al., 1987; Donoghue & Doyle, 1989; Loconte studies have been restricted to single families (Doe- & Stevenson, 1991; Martin & Dowd, 1991; Ham- bley et al., 1990; Kim et al., 1992) or putatively by & Zimmer, 1992; Hufford, 1992; Olmstead et closely related families (Soltis et al., 1990; Les et al., 1992; Taylor & Hickey, 1992). Such cladistic al., 1991; Donoghue et al., 1992; Olmstead et al., studies have previously been limited in scope; some 1992; Rettig et al., 1992). Most of the latter data matrices contain significant taxonomic gaps, studies began the process of incorporating signifi- and in others characters for some taxa are missing. cantly greater sampling to enhance their phylo- Both of these factors may have unpredictably mis- genetic perspective. The use of rbcL was spurred leading effects (Nixon & Davis, 1991; Platnick et by G. Zurawski, who generously made available a al., 1991). Despite a great deal of investigation set of internal sequencing primers. The advent of and analysis, seed-plant phylogenetics is, at best, temperature cyclers and high-temperature-resis- in a preliminary stage of investigation and knowl- tant DNA polymerases (sometimes termed Poly- edge. merase Chain Reaction or PCR) has greatly en- Molecular data, specifically DNA sequences, hanced rates at which gene sequence data are have received a great deal of attention as a potential accumulating, so that effects on phylogenetic es- source of "phylogenetically informative" charac- timates of more intensive sampling of sequence ters that are putatively less ambiguous than non- variation can now be investigated. molecular characters. Such pronouncements suffer We wish to examine here the degree to which from the limitation that, at higher taxonomic levels, a representative sampling of sequence variation for no extensive sampling and phylogenetic description rbcL contains evidence of the evolutionary history of DNA sequence variation has taken place. The of seed plants. In this study, we address the quality most taxonomically comprehensive analysis of nu- of evidence present in rbcL sequences for all major cleic acid sequences published so far on plants seed-plant lineages (roughly 265 families, the exact (rRNA; Hamby & Zimmer, 1992) used only 60 number depending on the taxonomic scheme fol- taxa, and a number of these were partial sequences. lowed). To a limited extent, we will compare our Martin & Dowd (1991), using nucleic acid se- phylogenetic hypotheses with recent schemes, but quences of the small subunit of RuBisCO (rbcS) such comparisons are difficult and must be consid- inferred from amino acid sequences, studied 335 ered heuristic because sister-group relationships ex-

from the DGAPA, Universidad Nacional Aut6noma de M6xico, Mexico, to LEE. We also thank David L. Swofford and John M. Mercer for assistance and advice in running the parsimony analyses; William S. Alverson, Michael T. Clegg, Julie Dowd, E. Allen Herre, Peter G. Martin, and James E. Rodman, for access to their unpublished sequences; Jeffrey J. Doyle, Robert F. Thorne, Peter Westin, Michael J. Donoghue, and several anonymous reviewers for their helpful suggestions; and the Royal Botanic Gardens, Kew, for support in the final stages of the preparation of the manuscript. Footnotes 2-23 follow the Literature Cited. 530 Annals of the Missouri Botanical Garden

pressed in cladograms are difficult to relate directly Cornaceae, Ericales, Magnoliaceae, Zingiberanae), to diagrams and statements of progenitor/descen- whereas others are poorly sampled (e.g., dilleniid dant relationshipsused in many taxonomic schemes. orders, especially Violales and Theales). Despite Dahlgren et al. (1985) and Dahlgren & Bremer lack of a coordinated effort, all subclasses and (1985) have published analyses most similar to the orders have at least some representatives. All spe- ones presented here but they are not of a similar cies that are used in this issue of the Annals of scope. Although some specific topological compo- the Missouri Botanical Garden are listed alpha- nents can be compared to other cladistic studies betically by family in a final Appendix along with (Conti et al., 1993; Rodman et al., 1993; Qiu et other information concerning voucher status, se- al., 1993; and several of the other studies in this quence gaps, literature citations for published se- issue), morphological phylogenetic studies at this quences, and figures of this paper in which each level with similar, broad taxonomic sampling do taxon occurs. Some taxa that were included in the not exist. The computational difficulties of evalu- 475-taxon data set were excluded from the 499- ating internal support (e.g., the bootstrap, Felsen- taxon matrix. Thus, in the second analysis certain stein, 1985; decay analysis, Bremer, 1988) or families have fewer representatives, but the overall departure from matrix randomness (Archie, 1989; representation of lineages is greater. Faith & Cranston, 1991; Killersj6 et al., 1992) Due to the large number of laboratories that for such large numbers of taxa likewise prevent us contributed unpublished sequences, no standard- from addressing extensively these issues here. We ized procedure was used to produce the sequences do present an analysis of families traditionally re- analyzed here. A generalization would be the fol- ferred to Hamamelidae as an example of how in- lowing: a fragment containing rbcL was amplified ternal support for specific clades might be exam- from a total DNA extract using primers that flank ined. (See also other papers in this issue that address or are near the ends of the coding region; this internal and external support for subsets of the fragment was then directly sequenced using one of general results.) Thus, the broad relationships de- several different procedures or was cloned using scribed here can be used to focus more restricted standard recombinant DNA techniques; dideoxy (with fewer taxa), and therefore more rigorous, sequencing generally included both strands for at investigations. least 4/5 of the minimally 1428-bp gene. Some work- We expect that patterns presented here will ers used more closely spaced primers to sequence change somewhat as sequences of more species are only one strand of DNA; either strategy appears added or if methodological improvements permit to provide reasonably error-free sequences. Most exact solutions (for a discussion of progress, see extraction protocols relied on fresh or freshly dried Penny et al., 1992). These results nonetheless have leaf samples, but some samples were amplifiedfrom great value, both from heuristic and methodological DNA extracted from herbarium specimens as old perspectives, although the preliminary nature of as 20 years. these studies precludes a detailed examination of Amplification of rbcL from some taxa produced implications for seed-plant taxonomy and character two different products. Some of these products were evolution, as well as investigations of gene and different enough in size to observe on an agarose protein evolution. gel, whereas others were detected initially because We have generally followed the taxonomic cir- multiple bands occurred at the same points in au- cumscriptions of Cronquist (1981) for dicots be- toradiograms, indicating that more than one tem- cause this system conforms closely to those used plate was present. Nearly all cycads produced two in most textbooks and floras. For monocots, we loci (Hills & Chase, unpublished), which were sep- have adopted the system of Dahlgren et al. (1985) arately cloned and sequenced to characterize both but have changed the superorder ending "-iflorae" copies. In cycads examined, one copy contained used by Dahlgren to the more appropriate "-anae" deletions that disrupted the reading frame, an in- (Thorne, 1992). dication that this copy may represent a "pseudo- gene." In Convolvulaceae, Olmstead (unpublished) detected two copies of rbcL, one copy of which contained deletions. In Canella (Canellaceae; Qiu MATERIALS AND METHODS et al., 1993) and Galphimia (Malpighiaceae; M. Selection of taxa for this study was not guided W. Chase, H. G. Hills & W. R. Anderson, un- by a specific plan. Close examination of genera published), two size-conserved copies of rbcL were included in the analysis will reveal an uneven tax- also encountered. In Galphimia, one copy clearly onomic distribution; some groups are well repre- contained substitutions at numerous sites otherwise sented (e.g., Asteraceae and Asteridae in general, conserved among angiosperms, suggesting that this Volume 80, Number 3 Chase et al. 531 1993 Phylogenetics of Seed Plants

copy was a pseudogene. In Canella, no such un- Although the assumptions of the Albert et al. model usual substitutions were observed in either copy. are admittedly simplistic, we nonetheless support Two, reading-frame-intact copies of rbcL are also the investigation of weighting approaches to nu- reported in Ulmaceae (E. Conti & K. J. Sytsma, cleotide data and view the model used here as a unpublished). For Canella, we included both se- justifiable first approximation. It must be recog- quences in all analyses, but because the two se- nized that giving all categories of molecular change quences are always each other's sister we show equal transformational weight is also an assump- only the position of "Canella" (in fact, there are tion, but one that the investigation of Albert et al. two terminals here; the complete matrix used in (1993) found to be adequate if sampling effects Search II thus has 500 terminals but only 499 were not a factor. Actual weights applied in this taxa). study fall within such a narrow and minimally Sequences of rbcL were easily aligned by sight. asymmetrical range that deviation of results from Among land plants, the coding region contains little those using the "equal weighting" criterion of Fitch size variation through base position 1428 (num- (197 1) is likely to be slight (see Albert et al., 1993). bered from the first nucleotide of methionine in the Recently, concern has focused on the probability start codon, AUG). Positions 1426, 1427, and that "islands" of equally parsimonious trees exist, 1428 are the most common stop-codon among land particularly in large data sets (Maddison, 1991). plants; longer reading frames, up to 1452 in some Because of their enormous size, our analyses do monocots and 1458 in the Asteraceae (Kim et al., not use methods of multiple random taxon-addition, 1992), appear to be due to insertions, often of a which have been suggested to uncover such dis- short repeating sequence. Most laboratories have junct, equally optimal islands. This topic is ad- sequenced past this codon, but for phylogenetic dressed in most empirical papers in this number. analysis we have terminated all sequences at po- The search for parsimonious trees consisted of sition 1428 to be confident that we have analyzed several separate but linked heuristic searches using homologous regions (the portion of the gene in- either PAUP 3.Or (Search I; 475 taxa) or 3.Os cluded in this analysis for each species is also (Search II; 499 taxa). All searches included the included in the Appendix). All sequences were en- full data matrix (all codon positions). Search I was tered into a text file in NEXUS format (used by performed on a SPARC II (Sun, Inc.) workstation PAUP 3.0; Swofford, 1991) as complete sequences (PAUP 3.0 for non-Macintosh computers is avail- and then analyzed directly in nucleotide form. Ma- able only by special arrangement with D. Swofford). trices used in both searches are available from the For Search II, a Macintosh Quadra 800 with 20 first author upon receipt of request and a diskette MB RAM was used. (Although slower than a Sun for each matrix. computer, the more interactive nature of a search on a PC is preferred by many workers.) In Search I, an initial heuristic search with character-state changes given equal weight (i.e., TREE-SEARCH STRATEGY "unordered" status), SIMPLE data addition se- Parsimony-based methods permit direct exam- quence, STEEPEST DESCENT, and NNI (nearest- ination of hypothesized character-state changes on neighbor interchange) branch swapping algorithm the reconstructed tree, and this information can was used to find a single tree (MULPARS option be used in studies of molecular evolution. (These deactivated). The second phase used this single are, however, likely to be underestimates of se- tree obtained from phase one as a starting tree for quence change and could be misleading for this another heuristic search, this time using the SPR reason.) Numerous empirical studies have shown (subtree pruning-regrafting) branch swapping al- that not all classes of substitutions are equally like- gorithm with MULPARS deactivated again. The ly, and this kind of information may be incorpo- third phase paralleled the first and second; the rated into weightng schemes for nucleotide data single SPR tree from phase two was used as the (Swofford & Olsen, 1990; Albert & Mishler, 1992; starting topology for a heuristic search using the among others). Various models of molecular evo- TBR (tree bisection-reconnection) branch swapping lution exist, and appropriately circumspect use of algorithm, again with MULPARS deactivated. these may assist in the separation of historical signal The fourth phase used the single TBR tree as from homoplasy. The character-state weighting the starting point for a heuristic search employing model of Albert et al. (1993, this issue) uses prob- a character-state weighting criterion (with a dif- ability formulae to calculate weights for different ferent step matrix for each codon position; cf. classes of molecular change. We have used their Swofford, 1991; Albert & Mishler, 1992; Albert method in the 475-taxon analysis presented here. et al., 1993). This time MULPARS was activated. 532 Annals of the Missouri Botanical Garden

Transversion substitutions were weighted over duced by using the CLOSEST addition sequence transitions differentially by codon position (Albert with the HOLD option set for five trees (this in et al., 1993). The specific weights used were the effect permitted initial swapping on several differ- following: for transitions, 5520 (first positions), ent starting topologies). Approximately 120 hours 6368 (second positions), 4039 (third positions); for were required merely to add all taxa in this manner. transversions, 6620 (first positions), 7470 (second The initial search (i) used NNI swapping and positions), 5127 (third positions). These weights STEEPEST DESCENT with MULPARS off. The were calculated from empirically derived param- shortest single tree found was then swapped on eters (see Albert et al., 1993, for a complete de- using (ii) TBR, which generally found a shorter scription and justification). Because use of step tree, at which time (iii) NNI (with MULPARS) was matrices is CPU-intensive, this search was exe- used. When use of MULPARS resulted in 3,900 cuted with the simplest branch-swappingalgorithm, trees, which used up available RAM, a single tree NNI. Additionally, because of dynamic RAM lim- was randomly selected (iv) to swap on with TBR itations (a tree of 475 terminals occupies a great (MULPARS off). If this resulted in a shorter tree deal of memory), we restricted our search to a being found, the search was then stopped and re- maximum of 500 trees. Although the initial TBR started (iii, again) using NNI and MULPARS and tree was by default a member of a single island, this shorter tree as starting point (iii and iv were we hoped to provide a bridge to shorter trees through repeated until no shorter trees were found). The use of the STEEPEST DESCENT option (see Mad- shortest tree length found with this method was dison, 1991; Swofford, 1991). This final step yield- 16,305. Three randomly selected trees from the ed 500 equally parsimonious, weighted trees. Be- 3,900 saved at this length were swapped in suc- cause the maximum prespecified number of trees cession to completion with TBR (no MULPARS), was found, many others probably exist at the same and no shorter trees were found. A strict consensus length. Search I required approximately 200 hours tree was computed, and branch lengths for one to complete. randomly selected tree were calculated using the Search II was performed for three reasons. ACCTRAN optimization. Search II thus used no First, the likely existence of other islands of equal relative weighting; it required approximately four or greater parsimony prompted us to use a strategy weeks to complete. that would be more likely to find shorter trees and perhaps a different topology. Second, we were con- CAVEATS cerned about the effects of the Albert et al. (1993) weighting scheme upon the resulting topology. Methodologically, these searches suffer from (i) Third, we wished to examine positions of additional uncertainty about maximum parsimony, (ii) un- taxa (and make use of updated sequences) that questionable absence of many trees at the same became available after Search I was completed; level of optimality, (iii) identification of only a single many of these belonged to previously unrepre- topology, and (iv), in Search I, incomplete branch sented lineages. Differences between these two sets swapping on any of the shortest trees found. We of trees could thus be due to different taxon sam- would never recommend these search strategies for pling, corrections or completions of sequences after smaller data sets, but several options were seriously Search I was finished, or search strategy. We did restricted by the number of taxa included (these not intend these two searches to be controlled, are reputedly the largest PAUP analyses attempted direct tests (i.e., with only one variable differing to date). Specific sections of the general topologies, between them); we show them both, rather than when analyzed in a more "localized" manner, pro- simply the one that we judge to be better (Search vide different sets of relationships (see Michaels et II), because their similarities, despite variation in al., 1993; Morgan & Soltis, 1993; both this issue). taxon composition and search strategy, are con- The broader taxon distributionof the general anal- siderable. They each represent results of searches ysis (thus with far greater outgroup information) that in their own context are worthy of publication, may be assessing character-state change on the and their differences should be viewed as reasons immediate branch leading to a specific ingroup to be skeptical of both and as cause for future differently than in more restricted analyses. Great- study with more rbcL data as well as other char- er outgroup information could "improve" the in- acters. group analysis or add spuriousness to it; it is gen- In Search II, we were able to save more trees erally best to implement tree searches both with at the shortest length found: 3,900 rather than and without outgroups to examine their effects. If only 500 trees. The initial starting tree was pro- more restricted analyses differ from those pre- Volume 80, Number 3 Chase et al. 533 1993 Phylogenetics of Seed Plants

sented here, we would certainly favor the former pendix). Many tissue samples were provided by because of increased confidence in finding parsi- botanical gardens, and, if vouchers are not included monious solutions. We nonetheless view these anal- with samples, investigators are dependent upon yses as instructive about seed-plant relationships identification of these plants by the respective or- and the utility and limits of rbcL information (see ganizations (see Goldblatt et al., 1992; it is critical Discussion). that a voucher sample taken from the same plant The major limiting factor in our studies is clearly used for DNA extraction be included at the time matrix size. When confronted with the largest mo- of collection; accession numbers or vouchers taken lecular database relevant to seed plants, compu- previously are subject to later events, such as lost tational trade-offs inevitably arose. The amount of labels or collector mistakes). Because a number of time spent on these approximations may not be the samples used in these two analyses are un- directly proportional to the time taken to generate vouchered (see Appendix), reproducibility is com- all the rbcL sequences, but no method exists to promised. In cases with multiple species from a predict how many trees at how many steps shorter family, we should be able to recognize grossly mis- might have been found after additional months or identified samples (but this is true for only 37% of even years of continuous computation. We have the 265 families represented). opted for the approximationspresented here rather than commit ourselves to an open-ended experi- RESULTS ment. Potential effects of errors in autoradiogramread- For display purposes, we show here the com- ing and data entry should be considered. We de- binable component consensus (Bremer, 1990) of tected a number of internal stop-codons in se- the 500 equally parsimonioustrees found in Search quences used in these analyses, and other workers I (Figs. 1-15, A series). Because these are char- have reported errors of various kinds. Most of these acter-state weighted trees, tree lengths and tree were corrected in the matrix used in Search II. statistics (e.g., consistency index, etc.) are not com- Certainly the potential for errors is present, but parable to those of Fitch trees and are not given the effects of such mistakes should not be extensive here. For Fitch trees found in Search II, the length because they are likely to be random. was 16,305 with a consistency index (C.I., ex- Identification of some taxa in this analysis has cluding unique substitutions and constant charac- been questioned due to peculiarity of placement. ters) of 0.102 and a retention index (R.I.) of 0.63 2. One example of this has been now identified among The branch lengths, again with ACCTRAN opti- the species used in Search I. We have not expunged mization, are shown on one of the 3,900 trees it from the illustrations; the results represent the selected at random (Figs. 1- 15, series B). Branches outcome of real tree searches and are instructive that collapse in the strict consensus tree are indi- for that reason. The sequence analyzed was an cated by arrows on the B series (Figs. 3-15B). actual member of the clade into which it was placed, First we will summarize the topology found in but it was not the species to which it had been Search I ("A" series of figures). The results of attributed. The material sent by a botanical garden Search II ("B" series) have been interdigitated with and labeled as Kirengeshoma was evidently mis- those of Search I to facilitate comparisons. After identified. It was almost certainly a member of the describing the results of Search I, we briefly ex- Parnassia group (Saxifragaceae sensu lato; the amine major differences between the two results. position it occupies in Fig. 1lA) rather than a Note that all figures in the A series are from the member of Hydrangeaceae. Sequencing of another combinable component consensus tree, whereas in specimen of Kirengeshoma and subsequent data the B series, Figures 1B and 2B are the strict analysis revealed the expected placement of this consensus tree (branch lengths in Fig. 2B, however, genus with other Hydrangeaceae (Xiang & Soltis, were taken from a single tree) and the remainder unpublished). Other "surprising" results also have (Figs. 3B- 15B) are a single tree randomly selected been checked by obtaining another sample of the from 3,900 equally parsimonious Fitch trees (this taxon in question and re-sequencing rbcL. The may be confusing; for example, whereas the po- original sequence of purportedly saxifragaceous lytomy among monosulcate clades in Figs. 1B and Montinia (which nested in Solanales) was checked 2B is due to variation among the 3,900 trees for and found to be accurate. Still others (such as branches in this portion, the topology shown in 3B Sargentodoxa among Fabaceae) are still being is resolved because it is resolved in the single tree reassessed. Most workers have tried to make selected). To indicate branches of the B series that vouchers for each species in this study (see Ap- are absent in the strict consensus tree of the 3,900, 534 Annals of the Missouri Botanical Garden

we have placed arrows to their right. The branch noliales, (ii) Laurales, and (iii) "paleoherbs" (here lengths presented in the B series should under no defined as composed of Aristolochiales, Piperales, circumstances be interpreted as meaningful mea- and Nymphaeales; Fig. 4A). Monocots (also with sures of support; thus, in the example of Hama- uniaperturate pollen and sometimes included in melidae provided, branches that decay at one step "paleoherbs"; Donoghue & Doyle, 1989) repre- less parsimonious have lengths that range from 2 sent a fourth member of this lade. Among the to 16 steps, whereas those that decay at four steps paleoherbs are also nested several problematic fam- less parsimonious range from 7 to 14 steps (Fig. ilies: Illiciaceae, Schisandraceae (both Illiciales), 16). The sole reason for providing branch lengths Amborellaceae (Laurales), and Austrobaileyaceae in the B series is to permit readers to estimate (Magnoliales). Chloranthaceae are also allied cla- roughly relative degrees of divergence and to iden- distically with the paleoherbs, but Chloranthus does tify cases in which long terminal branches are not form a monophyletic group with other Piper- connected to short internal branches (a situation ales. in which adding related taxa often radically alters Monocots are a well supported monophyletic hypothesized relationships). group (see Duvall et al., 1993, and Qiu et al., Search I. Unless stated otherwise, we have 1993, both this issue) and are derived from within used the taxonomic circumscriptions of Cronquist monosulcate Magnoliidae; the paleoherbs are their (1981) for dicots and Dahlgren et al. (1985) for immediate sister group (Fig. 4A). Within monocots monocots, although we acknowledge that other re- (Fig. 5A, B), Aranae plus Pleea (of polyphyletic cent systems fit these results better. We arranged Melanthiaceae; Lilianae) are basal-most, followed the unrooted trees of both searches with cycads by Alismatanae plus Burmannia (Burmanniaceae) sister to all other seed plants (Figs. 1-3) in accord and Aletris (Melanthiaceae). Lilianae form a para- with recent results of several non-molecular cla- phyletic series of three lineages that correspond distic studies (Crane, 1985, 1988; Doyle & Don- well to Dioscoreales, Liliales, and Asparagales of oghue, 1986, 1992). In Search I, conifers are Dahlgren et al. (1985), except in the placement paraphyletic, but some trees (not shown) found in of certain families (Iridaceae, Orchidaceae, and Search II have a monophyletic conifer lineage; the Smilacaceae) and genera (Chamaelirium of Melan- strict consensus tree from Search II is unresolved thiaceae: Melanthiales). Vellozia (Velloziaceae: regarding conifers (Figs. 1B, 2B; we have cited in Bromelianae), Freycinetia (Pandanaceae: Panda- this section the B series of figures along with the nanae), and Sphaeradenia (Cyclanthaceae: Cy- A series if they include the same general set of clanthanae) together form a monophyletic lade taxa). The three genera of Gnetales, Ephedra, that is collectively sister of the Liliales. The "com- Welwitschia, and Gnetum, are highly divergent melinoid" group of monocots (Fig. 6A, B) incor- from all other seed plants but were nonetheless porates all of those that Harris & Hartley (1980) identified as sister of the angiosperms (Fig. 3A, B), found to exhibit fluorescing cell-wall phenolics. In within which Ceratophyllum (Ceratophyllaceae) both searches, this commelinoid lade includes alone is sister to and highly divergent from the rest monophyletic Arecanae and Zingiberanae and (Fig. 4A, B). The major feature of flowering plants polyphyletic Bromelianae and Commelinanae (Fig. (exclusive of Ceratophyllum) is their separation 6A, B). Cyclanthaceae have the same phenolic into two major groups; these correspond well with biosynthetic pathway but do not accumulate end distributionsof the two major angiospermous pollen products; they are not members of the commelinoid types, uniaperturate (monosulcate and monosul- assemblage (Clark et al., in prep.). cate-derived) and triaperturate (tricolpate and tri- The two orders of Magnoliidae with triaperturate colpate-derived). Ceratophyllum has inaperturate pollen, Ranunculales and Papaverales, form a lade pollen (Cronquist, 1981). The major exception to that is sister to the rest of "eudicots" (Fig. 7A, this split is the presence of tricolpate pollen in B). The term "eudicot" has been variously defined Illiciaceae and Schisandraceae, which fall among in the literature, but we use it here to refer to all the monosulcate taxa (Fig. 4A, B; see Qiu et al., angiosperms with triaperturate or triaperturate-de- 1993, this issue). No morphological support for rived pollen (Donoghue & Doyle, 1989; Doyle & monophyly of the monosulcate clade has been rec- Holton, 1991). This is one of the best supported ognized in the literature (their pollen type exists clades among angiosperms (Qiu et al., 1993, this among nonflowering seed plants and thus must be issue). Two other basal clades within the eudicots considered plesiomorphic). (Fig. 7A, B) consist of some Hamamelidae (Troch- Three monophyletic lineages within uniapertur- odendraceae and Tetracentraceae) and Platana- ate magnoliids were identified, and these corre- ceae, Sabiaceae, Nelumbonaceae, and Proteaceae. spond closely, although not exactly, to (i) Mag- Within eudicots, two large sister clades are iden- Volume 80, Number 3 Chase et al. 535 1993 Phylogenetics of Seed Plants

tified, one that corresponds roughly to Asteridae included in Geraniaceae (see also Price & Palmer, and the other to Rosidae (Figs. 1, 2). Membership 1993, and Morgan & Soltis, 1993, this issue). in both lineages is considerably expanded with re- The tenuinucellate/sympetalous lade that ter- spect to their circumscription by Cronquist (1981), minates in Asteridae sensu Cronquist (1981) was although less so with respect to the circumscriptions also identified by Olmstead et al. (1992) in their of Dahlgren & Bremer (1985) and Thorne (1992). efforts to circumscribe subclass Asteridae using These two major clades (Fig. 2A, B) reflect the rbcL sequences. This study greatly expands upon division of eudicots into two major groups (Young their sampling and identifies as members of the & Watson, 1970): sympetalous/tenuinucellate and asterid lade two lineages of often polypetalous polypetalous/not tenuinucellate (asterids and ros- Rosidae (Figs. 2A, 12A): (i) Santalales plus Paeoni- ids, respectively, in our figures). The "crassinu- aceae and Gunneraceae and (ii) some families of cellate" condition actually consists of several dif- Cornales plus Hydrangeaceae (see Xiang et al., ferent states. Exceptions to this generalizationexist, 1993, this issue). The sister group of Asteridae is but these traits appear much less homoplastic here a lade (Figs. 2A, B, 13A, B; see Kron & Chase, than in most systems of classification. The basal- 1993, this issue) that contains the dilleniid orders most lineage within Rosidae includes Saxifragaceae Ebenales, Ericales, Primulales, Diapensiales, plus sensu stricto and Crassulaceae besides lower ha- some members of Theales (Actinidiaceae and Thea- mamelids such as Cercidiphyllum and Hamamelis ceae). Sarraceniaceae (Nepenthales; Dilleniidae)and (Fig. 8A; see Morgan & Soltis, 1993, this issue, Roridula (but not Byblis of Byblidaceae of Ro- for a discussion of Saxifragaceae sensu lato). The sales: Rosidae) are also members of this lineage next-most-basal group contains Caryophyllidae (in- (Albert et al., 1992b). Polemoniaceae (Solanales: cluding Plumbaginaceae and Polygonaceae; Rettig Asteridae) and Balsaminaceae (Geraniales: Rosi- et al., 1992) plus Droseraceae, Nepenthaceae (Ne- dae) also belong to this ericalean/ebenalean group. penthales, Dilleniidae; see Albert et al., 1992b), (See also Olmstead et al., 1993, this issue, for a Dilleniaceae (Dilleniidae), and Vitaceae (Fig. 9A). treatment of the Asteridae sensu lato.) The remaining Rosidae are split into two large Asteridae sensu Cronquist split into two major sister groups (Figs. 2A, I0A, lIA). In one (rosid sister groups. In one of these (asterid II; Fig. 14A, I) are several families of higher Hamamelidae, Eu- B) are families of Asterales, Calycerales, Campan- phorbiales, Fabales, Linales, Polygalales, and Ro- ulales, Dipsacales, and some Solanales (Menyan- sales (Fig. 1 lA). Other members of this lade in- thaceae). Rosid taxa that are members of this lade clude a number of dilleniid families: Ochnaceae include Apiales, Aquifoliaceae (Celastrales), some (Theales), Datiscaceae, Passifloraceae, and Viola- Cornaceae (Cornales), Pittosporaceae and Gros- ceae (all Violales). Ordinal boundaries of this group sulariaceae (both Rosales). In the other major lade of Rosidae (sensu Cronquist, 1981) are largely (Fig. 1 5A, B) fall orders Callitrichales, Gentianales, unsupported; this assemblage is particularly het- Lamiales, Rubiales, Scrophulariales, and most So- erogeneous. The largest polytomy in the consensus lanales, although these ordinal limits are not always tree from Search I occurs at the base of this group, supported (see Olmstead et al., 1993, this issue). and sampling of the families that potentially belong In this lade (Fig. 15A, B) is a group that includes to this lade is the most sparse in this analysis. rosids Aucuba (Cornaceae: Cornales) and Garrya The other major lineage of Rosidae (rosid II; Fig. (Garryaceae: Cornales) and hamamelid Eucommia 1 A) includes orders Myrtales (see Conti et al., (Eucommiaceae: Eucommiales), all of which ac- 1993, this issue) and Sapindales, for which ordinal cumulate aucubin (Cronquist, 1981) and share dis- boundaries are reasonably intact. Malvaceae (Mal- tinctive anatomical wood characteristics (E. Whee- vales; Dilleniidae) and all but one of the mustard- ler, pers. comm.). Thus the suite of floral oil families (those in Capparales plus others in Dil- characteristics that have been interpreted as sup- leniidae; see Rodman et al., 1993, this issue) are port for the monophyly of Asteridae sensu Cron- associated with Sapindales. Geraniaceae are also quist (1981) appears either (i) to have twice arisen members of this lade, although other members of independently from ancestors with rosalean and Geraniales appear elsewhere (Oxalidaceae with a cornalean floral traits or (ii) to have undergone group of families in Rosales, Fig. llA; Balsami- reversals in groups traditionallyincluded in Rosidae naceae with Ebenales, Fig. 13A, B; and mustard- (sensu Cronquist;Donoghue et al., 1992; Olmstead oil-producing Limnanthaceae and Tropaeolaceae et al., 1992, 1993, this issue). with Capparales, Fig. I1A, B; Price & Palmer, Search II. A number of taxa from Search I 1993, this issue). Two members of Rosales, Greyia were removed from Search II (all marked with a (Greyiaceae) and Francoa (Saxifragaceae), appear "t" in the A series of figures) to accommodate the derived from within Geraniaceae, if Viviania is representatives of additional lineages in Search II. 536 Annals of the Missouri Botanical Garden

Most of the removed taxa were from monophyletic noliid species added in Search II represent addi- families in which six or more species were present tional members of families already present in Search in Search I (for example, Asteraceae, Ericaceae sen- I, and all of these form monophyletic units with su lato, Magnoliaceae, and Poaceae). Two others, other members of their respective families (Fig. Burmannia (Burmanniaceae) and Hydrolea (Hy- 4B). drophyllaceae), were removed because they are Sister to all other monocots is Acorus (Araceae; highly sequence divergent from all other taxa; in see Duvall et al., 1993, this issue). Calochortus separate, smaller analyses, these two appear to be (Calochortaceae) is sister to Liliaceae, Hemero- involved in "branch attractions" and attach in rad- callis (Hemerocallidaceae) is sister to Chlorophy- ically different positions as other taxa are added tum (Anthericaceae), and Lomandra (Dasypogona- or removed (other similarly divergent genera, for ceae) is a member of the often arborescent lade example, Paeonia and Gunnera, do not cause these composed of Agavaceae, Asphodelaceae, and Xan- problems and were kept; we admit to being rela- thorrhoeacae within Lilianae (Fig. 5B). Additional tively arbitrary in removing only these two taxa). members of families in Search I are all placed as Burmannia presents an interesting case. Members sister taxa to their respective family representa- of Burmanniaceae are often achlorophyllous; in tives. Among the commelinoid lade identified in spite of being green, Burmannia may still derive Search I (Fig. 6A, B) are the following additional a great deal of its nutrition through its mycorrhizal families: Sparganium (Sparganiaceae) sister to Ty- associate. In such cases in other families, a number pha (Typhaceae) and Lachnocaulon (Eriocaula- of protein loci exhibit higher rates of sequence ceae) among the graminoids. divergence (as measured by relative branch lengths Among eudicots (Fig. 7A, B), Pachysandra when compared to completely autotrophic mem- (Buxaceae) is sister to Trochodendrales (Trocho- bers of their lineages; C. dePamphilis, pers. comm.). dendron and Tetracentron). Composition of the Thus the high levels of sequence divergence, which various rosid clades is somewhat different in Search make it difficult to place these taxa accurately, are II (Fig. 2B; see below), but the following additional a product of or at least associated with their partial taxa are placed roughly among the rosid II (Fig. heterotrophy. 1OB) lade identified in Search I: Shorea (Dipter- For Search II, additional taxa were available ocarpaceae), Theobroma (Sterculiaceae), Tilia (Til- (these species are marked with asterisks in the B iaceae), and Bombax (Bombacaeae) are members series of figures). The placements of these are of a malvalean lade (represented only by Gos- described first, and then different arrangements of sypium in Search I). Akania (Akaniaceae) and taxa included in both searches are identified (the Bretschneidera (Bretschneideraceae), both Sap- clades involved with major shifts of position are indales, are members of the mustard-oil lade. Three marked with an "?" in Fig. 2B). This last section additional members of Capparaceae, Cleome, Koe- describes only the major shifts of position, but many berlinia, and Setchellanthus, along with Cappa- "minor" shifts also occur within clades (for ex- ris, create a polyphyletic Capparaceae (Fig. 1OB; ample, 12 "minor" shifts take place among the see also Rodman et al., 1993, this issue). Hyp- taxa in Fig. 15, a lade in which only a few new seocharis (Oxalidaceae) is sister to a lade con- species were added). What constitutes a "major" taining many Geraniaceae (Fig. 1OB; see Price & versus a "minor" alteration is, of course, a matter Palmer, 1993, this issue). Among members of rosid of personal perspective. We would therefore advise I (Fig. 1I B, C) are representatives of the following readers to examine carefully the trees from both new families: Reinwardtia (Linaceae) is sister to searches for taxa of specific interest, which is one Viola (Violaceae); Sargentodoxa (Sargentodoxa- reason we presented and intercalated the results ceae) is imbedded in Fabaceae; Humulus (Can- of both analyses. nabinaceae), Trema (Ulmaceae), and Boehmeria Positions of additional taxa. Ginkgo (Gink- (Urticaceae) fall into an urticalean lade with Mo- goaceae) intercalates between cycads and conifers rus and Ficus (both Moraceae), but Ulmaceae are (Figs. 1-3B). Taxus (Taxaceae), Cephalotaxus paraphyletic to Cannabaceae; Coriaria (Coriaria- (Cephalotaxaceae), and Sciadopitys (Taxodiaceae caeae), Begonia (Begoniaceae), and three genera or in its own family) are members of the non- of Cucurbitaceae are placed with Datiscaceae; Bet- Pinaceae lade of conifers, with Sciadopitys sister ula (Betulaceae) and Carya (Juglandaceae) are to the rest of that lade (Fig. 3B). Within magnoliid members of the lade containing Fagaceae and angiosperms, Lactoris (Lactoridaceae) is sister to Casuarinaceae; Mouriri and Osbeckia (Melasto- Aristolochia (Aristolochiaceae). Other new mag- mataceae), Punica (Punicaceae), Trapa (Trapa- Volume 80, Number 3 Chase et al. 537 1993 Phylogenetics of Seed Plants

ceae), and Heteropyxis (Myrtaceae) are members Families of Myrtales (plus Qualea of Vochysiaceae, of a lade of (mostly Myrtales) that includes On- Polygalales) and the lade containing Viviania, agraceae and Combretaceae (Fig. 1 IC). In asterid Wendtia (both Geraniaceae), Greyia (Greyiaceae), lineages, two additional families have been added: and Francoa (Saxifragaceae) are shifted from rosid Byblis (Byblidaceae) and Vahlia (Saxifragaceae) II to rosid I (Fig. 1 iB, C), and relationships of are placed among Schrophulariales (Fig. 15B). intermediate-level clades (containing several fam- Changes in placements of major clades. At ilies, i.e., those of Urticales, Fagales, etc.) are higher levels among the seed plants, Search II somewhat modified from their position in Search I. produced relatively few "major" differences in tax- The third shift involves the two taxa that were on placements from the topology of Search I. The sister to the expanded caryophyllid lade from conifers in some, but not all, 3,900 trees are mono- Search I (Fig. 9A): Dillenia (Dilleniaceae) and Vitis phyletic. Piperales-Aristolochiales were sister to (Vitaceae), which become sister to the rest of the all monosulcate angiosperms (Figs. 2B, 4B). The larger asterid lade (Fig. 12B; Vitis and Dillenia "paleoherb" lade of Search I (Fig. 4A) was thus in a sense exchanged positions with Santalales). split in two with the portion containing Chloran- Again, these groups have short internal branches, thaceae, Illiciales, Austrobaileyaceae, Amborella- and these shifts would require little change of over- ceae, and Nymphaeales situated as sister to Mag- all parsimony. noliales. Although these may seem to represent In several instances, the topology of Search II major shifts, extremely short branches separate (which we favor because it was a more complete these clades (Fig. 4B); thus neither topology has search) is more similar to that found in investi- much internal support (Qiu et al., 1993, this issue). gations of restricted nature (for example, outgroup Within monocots, only shifts among the groups in relationships of Asteraceae are identical with those Aranae, Alismatanae, and Liianae occurred (Fig. found by Michaels et al., 1993, this issue, and 5B): Aranae (minus Acorus) are sister to Alisma- Olmstead et al., 1992, whereas those of Search I tanae; the lade containing Pandanaceae, Cyclan- deviated in several ways). thaceae, and Velloziaceae is isolated and no longer sister to Liiales, and Dioscoreales are sister to the DISCUSSION commelinoid taxa (the position of Asparagales in the trees from Search I). Among commelinoid taxa Although rbcL sequences for several groups of (Fig. 6B), Stegolepis (Rapateaceae) is sister to spore-bearing plants are available (true mosses, Bromeliaceae, Typhales are sister to Juncaceae- hornworts, liverworts, Equisetum, Isoetes, Lyco- Cyperaceae, and Flagellaria is sister to the other podium, Psilotum, and both eu- and leptosporangi- graminoid lade. ate ferns), their use as outgroups is complicated Among eudicots, three major shifts of taxa pres- by extensive sequence divergence relative to that ent in Search I occurred. The first series of re- in the seed-plant ingroup. No other extant lineages arrangements involves the heterogeneous asterid of land plants are likely to have shared a common V lade (Fig. 12A). Gunnera (Gunneraceae) in- ancestor with seed plants for well over 350 million tercalates as an isolated lineage (Fig. 7B) between years, and a great deal of sequence change, much Trochodendrales (plus Pachysandra of Buxaceae) of it in the form of multiple, unrecoverable sub- and higher eudicots (asterids, caryophyllids, and stitutions, has occurred. Analysis of these other rosids). Santalales, Phoradendron (Viscaceae), land plant sequences produces topologies (not shown, Schoepfia (Olacaceae), and Osyris (Santalaceae) but see Hamby & Zimmer, 1992, for similar re- become sister to Caryophyllidae-Droseraceae- sults) that are radically different from all previous Nepenthaceae (Fig. 9B), and this larger caryophyl- hypotheses of relationships (e.g., Bremer et al., lid lade is shifted from a position within the rosid 1987). In contrast, seed-plant relationships pre- lade (rosid III; Figs. 2A, 9A) to sister to the larger sented here are at least congruent in gross aspect asterid-rosid lade (Figs. 2B, 9B). The remaining with comparable morphological studies (Crane, member of asterid V, Paeonia (Paeoniaceae), is 1985, 1988; Doyle & Donoghue, 1986, 1992; deeply imbedded within rosid III lade (Fig. 8B) Loconte & Stevenson, 1991). Addition of highly as sister to Crassulaceae. We attach little signifi- sequence-divergent outgroups could be expected cance to these shifts of position; internal branches to increase ingroup homoplasy with unpredictable of these groups are among the shortest supporting topological results (Felsenstein, 1978). In these positions of major clades. analyses, we have chosen to use the more conser- The second series of shifts occurs within rosids. vative approach of an unrooted ingroup analysis 538 Annals of the Missouri Botanical Garden

of seed plants, which are almost certainly mono- and ribosomal studies (Hamby & Zimmer, 1992) phyletic (Doyle & Donoghue, 1986, 1992). are quite similar to ours as well. How these larger Likewise, effects of missing groups upon topol- groupings (clusters of families and in some cases ogies may be profound (Donoghue et al., 1989) orders) are inter-related is the point at which their and unpredictable, and we expect the absence of similarity diverges. Although having quite different the numerous extinct lineages of early land plants implications for angiosperm origins and evolution, in molecular data matrices to pose potentially se- the preferred hypothesis in one of these studies is rious problems for elucidating relationships of ex- not vastly different in relative parsimony from those tant lineages. Perhaps more conservative genes favored in other investigations. For example, with sampled for more of the sequence variation present mostly morphological data, Doyle & Donoghue within extant groups may be able to "bridge" gaps (1986) discovered a "paleoherb rooting" at one caused by extinction. Features of genome organi- step less parsimonious. Constraining a paleoherb zation (such as gene and intron content and gene rooting for angiosperms, at Nymphaeales (as in order; Downie & Palmer, 1992; Raubeson & Jan- Hamby & Zimmer, 1992), was also only slightly sen, 1992) may offer more robust hypotheses than less parsimonious in the subset of angiosperm rbcL gene sequences for such questions, but these are sequences studied by Qiu et al. (1993, this issue). likely to be too few to provide a fully resolved tree When examined to address basal angiosperm re- by themselves. A great deal more experimentation lationships, all these data appear to lack a strong with combined data sets of morphological and mo- historical signal. lecular characters is obviously needed, and a num- A number of phenomena have been suggested ber of these studies are underway (both with rbcL to be capable of confounding molecular phyloge- and rRNA/rDNA data). Consideration of these netic studies. We consider below several of these problems here is premature. factors and examine their potential to affect studies Sister-group status of angiosperms and Gnetales of rbcL sequence variation and then address some (Figs. 2A, B and 3A, B) is corroborated by other additional concerns about future directions of mo- cladistic studies (Crane, 1985, 1988; Doyle & lecular systematic study. Donoghue, 1986, 1992; Loconte & Stevenson, 1991). The isolated position of Ceratophyllum as EFFECTS OF PARALLELNUCLEOTIDE sister to all other angiosperms has been argued SUBSTITUTIONS IN INDEPENDENT LINEAGES previously (Les, 1988; Les et al., 1991). In studies An effect of unrecoverable (due to extinction) by Qiu et al. (1993, this issue) in which non- or unobserved (due to insufficient sampling) char- flowering seed plants were removed, this arrange- acter-state changes is the introduction of spurious ment was made equivocal by the existence of an- similarities, which may result in treatment of in- other equally parsimonious island in which Cera- dependently derived nucleotides at a given base tophyllum occurred in a radically different position. position as homologous (see Albert & Mishler, Hamby & Zimmer (1992; rRNA) also found a yet 1992). Such mistaken interpretations of indepen- different placement for Ceratophyllum (but we dent events can lead to "branch attractions" if an suspect that the sparser sampling of their study analysis includes a great number of such assess- may be responsible for most of the differences from ments (Felsenstein, 1978). Adequate taxon sam- those found with rbcL). In instances in which a pling, sometimes referred to as appropriate "taxon taxon's morphology and anatomy are as divergent density," is one means of reducing potentially in- and potentially modified as those of Ceratophyl- accurate assessments of similarity, but determining lum, its position becomes difficult to address ade- at what point sampling is sufficient has so far only quately in cladistic studies. Ceratophyllum has been been addressed in an a posteriori manner. absent from many morphological cladistic studies, The improvement afforded to assessments of such as those of Doyle & Donoghue (1986, 1992), character-state change by increased taxon sam- so corroboration is currently precluded. pling is counterbalanced by a decrease in com- The general groupings of angiosperms (exclusive putational speed and ability to ascertain how near of Ceratophyllum) identified in these two analyses results are to maximum parsimony. Intrafamilial are highly similar to each other and to those of studies are not as likely to be affected severely by most recent taxonomic schemes, particularly those these problems because, in general, numbers of of Dahlgren (1980), Dahlgren et al. (1985), and taxa are not as great and evenness of sampling is Thorne (1992; this last has admittedly incorporated better. At higher taxonomic levels within seed plants, results of several molecular investigations). Fur- a paradoxical impediment to progress arises: if thermore, results of rbcS (Martin & Dowd, 1991) taxon number is great enough to assess character- Volume 80, Number 3 Chase et al. 539 1993 Phylogenetics of Seed Plants

state changes accurately, then one reasonably can pled with manageable numbers of taxa (from the expect to reach only suboptimal phylogenetic so- standpoint of computation of minimal trees) and if lutions, but if taxon number is restricted enough sampling with sufficient numbers of taxa precludes to gain confidence of the maximum parsimony of assessing the parsimony of results, then we have the trees found, spurious assessments of character- reached an impasse until improved methods of anal- state identity could obscure all but the closest re- ysis are developed. An appreciation of this problem lationships. has led us to be skeptical of the overall topologies To evaluate relationshipsof Plumbaginaceae and presented (Figs. 1, 2), and competing ideas of re- Polygonaceae to families of Caryophyllales, Gian- lationships should not be overlooked when per- nasi et al. (1992) used a number of phenetic and forming more restricted analyses of these and other "phylogenetic" methods, including a Fitch-Mar- gene sequences. goliash dendrogram based upon genetic distances (Kimura, 1981; Felsenstein, 1990), maximum par- simony (PHYLIP, Felsenstein, 1990; PAUP, Swof- INTERNAL SUPPORT FOR THE BROAD TOPOLOGY: AN AD ford, 1991), and maximum likelihood (ML; Fel- HOC ANALYSIS OF SUBCLASS HAMAMELIDAE senstein, 1981). All three methods produced the For purposes of suggesting an appropriate man- same lack of resolution concerning relationships of ner to examine internal support of families tradi- these families; to state that Plumbaginaceae and tionally recognized as a natural group, we selected Polygonaceae are "not closely related" (meaning one of the most controversial subclasses, Hama- "closely similar") to Caryophyllales does not pre- melidae sensu Cronquist(1981), for which we have clude them nonetheless from being closest relatives. data from 18 of 24 families. Numerous phyloge- From the perspective of results presented here (Fig. netic and systematic studies of the Hamamelidae 9A, B), spurious similarities in the data analyzed have been completed (see various authors in Crane by Giannasi et al. (1992) to affect appear equally & Blackmore, 1989). The morphological features results of all three tree-building methods: Gossyp- suggesting a close relationship among these families ium simultaneously attracts higher asterids and are largely those associated with the temperate magnoliids (in our trees Gossypium is well imbed- amentiferous syndrome, and these clearly could be ded among rosids; rosid II, Fig. 1iA, B). (See the result of parallel modification in Olmstead et al., 1992, 1993, this issue, for an unrelated lin- eages. In their developmental example and discussion of the effects of taxon characteristics and wood anatomy, families of sampling.) Hamamelidae are par- ticularly heterogeneous (Cronquist, 1981; Crane Prospects for improvements of tree-building & Blackmore, 1989). In performing the broad methods with greater numbers of taxa exist (Penny analysis, we sought to avoid a priori ideas about et al., 1992). In the example of Giannasi et al. what constituted monophyletic subgroupings of an- none of (1992), the methods employed succeeded giosperms, but for this heuristic example we have in eliminating what we interpret as branch attrac- accepted ad hoc the outgroup relationships found tions due to the small number of taxa sampled and in the general study. use of distantly related outgroups. Phylogenetic We selected 72 species that included all mem- studies using morphological characters for a closely bers of Hamamelidae and their immediate sister related group of rosid or asterid families would taxa as identified in trees from both searches. No likely be affected adversely by outgroups of mag- attempt was made to select species that would noliids or monocots. This phenomenon is perhaps reproduce the particulars of the general topologies. even more probable with nucleotide data in which A tree search under the Fitch (equal weights) cri- homology is initially assessed only by nucleotide terion using 2,000 random sequence additions, position and character states are restricted to the MULPARS, STEEPEST DESCENT, and NNI same four alternatives. Character "homology" for branch-swapping (but permitting only 10 trees to distantly related taxa can be easily determined with be held at each step) found only one island at rbcL data (i.e., a given nucleotide position in this maximum parsimony (i.e., all trees could be found size-conserved gene; "primary homology," de by single branch swaps using any one of them as Pinna, 1991), but this does not mean that assess- a starting tree; cf. Maddison, 1991). Additional as ments of character-state homology (synapomor- well as shorter islands could still exist but are un- phy) are less subject to homoplasy than with other likely after 2,000 repetitions (this type of search data. required about 24 hours to complete on a Mac- If phylogenetic analyses of nucleotide data can- intosh Quadra 950 with 20 MB of RAM). After not be expected to reveal relationships when sam- random addition searches were completed, the trees 540 Annals of the Missouri Botanical Garden

found were used as starting points in a single anal- are shown to be grossly polyphyletic. Besides the ysis with MULPARS on and TBR swapping to amentiferous syndrome, the major trait of Ham- completion; this process should find all equally par- amelidae is the presence of tannins, which is like- simonious trees in the single island identified. Our wise compatible with a relationship to the other search found 36 equally parsimonious Fitch trees, tannin-containing families, Fabaceae, Rosaceae, three of which were optimal under the weighting Saxifragaceae sensu stricto, and Crassulaceae. criterion of Albert et al. (1993, this issue; see Many authors (e.g., in Crane & Blackmore, 1989) Materials and Methods). One of these was randomly have discussed these families in terms of which are selected and is shown with Fitch branch lengths "lower" and which are "higher" families. This (ACCTRAN optimization;Fig. 16). The tree length distinction finds some support from these results; was 2,234 steps, the C. I. was 0.288 (excluding the "lower" groups either stand in an isolated po- unique characters), and the R. I. was 0.532. The sition near the base of the eudicots (Eupteleaceae, maximum parsimony trees also were used to search Platanaceae, and Tetracentraceae-Trochodendra- for trees up to five steps less parsimonious under ceae) or basal within rosids (rosid IV, Fig. 2A; the Fitch criterion; the FILTER TREES option was Daphniphyllaceae, Hamamelidaceae, and Cercidi- used to identify trees at each length, and a strict phyllaceae), whereas most "higher" hamamelids consensus tree at each step was computed. The (Casuarinaceae,Fagaceae, Moracae, Ulmaceae, and number of steps less parsimonious at which each Urticaceae) demonstrate a well supported relation- topological component decayed was recorded (Fig. ship to Fabaceae or Rosaceae (d > 5 on two 16; "decay values" are shown below the branches: branches at the base of the largest lade in Fig. "dO" indicates that the branch is a polytomy in 16). The position of Leitneria in Sapindales near the strict consensus of the maximum parsimony Burseraceae is corroborated by a shared suite of trees, "dl" indicates that the branch is a polytomy secondary compounds and presence of intercellular in the consensus tree at one step less parsimonious, resin canals. etc.). The example presented above is not intended to This analysis identified eight lineages into which be more than a superficial phylogenetic treatment members of Hamamelidae fell (Fig. 16): Eucom- of families traditionally referred to Hamamelidae. mia, lade A, sister to Aucuba (Cornaceae) and It is meant to serve as an example of how the Garrya (Garryaceae) and nested within an asterid general topology, which itself is suspected of being lade; Hamamelidaceae, Cercidiphyllaceae, etc., suboptimal and presently cannot be examined by lade B, in a series paraphyletic to Saxifragaceae- decay analysis because of its size, may identify a Grossulariaceae; Fagales, etc., lade C, sister to relevant analysis within which questions of opti- Fabaceae-Polygalaceae; Urticales, lade D, sister mality and relative support can be addressed. We to Rosaceae; Leitneria, lade E, nested within fam- are pleased that general relationships found in the ilies of Sapindales; isolated Trochodendrales, lade broad analyses hold up well when addressed in this F, sister to Buxaceae; Platanus, lade G, situated and other more restricted investigations, none of in a heterogeneous group; and the last, Euptelea, which have found vastly different topologies. lade H, situated among Ranunculales. Many of these clades are well supported internally, decaying ON THE INFORMATIVENESSOF at three or more steps less parsimonious (some, ALL SUBSTITUTIONS such as Trochodendrales-Buxaceae and the Plat- The majority of character-state changes in pro- anus assemblage are weakly supported as mono- tein-coding genes have been demonstrated to occur phyletic lineages but clearly are not members of at third positions within codons, and numerous other well supported groups, leaving them in iso- empirical studies have shown third position substi- lated positions apart from other Hamamelidae). tutions to be more abundant in this and other data Results of this restricted analysis are congruent sets. Some workers have experimented with dis- with the topology found in the broad searches (Figs. carding third position substitutions from their anal- 1, 2) and indicate the level of internal support yses or analyzing nucleotide sequences inferred demonstrated by rbcL data. Future studies could from amino acid data (which standardizes all syn- combine morphological data with this molecular onymous substitutions; Martin & Dowd, 1991). In matrix and perform constraint experiments in which several studies (Conti et al., 1993, this issue; Don- various rearrangements of these taxa are examined oghue et al., 1992; Kim et al., 1992; Smith et al., for their relative degrees of parsimony. The general 1993, this issue), all three codon positions have conclusion from this example is that the Hama- been found to exhibit similar levels of homoplasy melidae do not form a monophyletic lineage; they (and perhaps similar rates of change per site as 590 Annals of the Missouri Botanical Garden

To gain a measure of the robustness of the as the "monosulcates" and those with tricolpate topology, decay of parsimony (Bremer, 1988) was and tricolpate-derived pollen types as the "eudi- examined. All maximum parsimony trees of both cots," the latter in accord with Doyle & Hotton's, islands were used as starting trees to search for 1991, use of that term). Five major lineages are trees up to five steps less parsimonious;the FILTER identified among the Magnoliidae; they are roughly TREES option was used to identify trees at each equal to the Magnoliales, Laurales, Piperales (in- length, and a strict consensus tree was computed cluding Aristolochiaceae and Lactoridaceae), Nym- at each step. The number of steps less parsimonious phaeales (excluding Nelumbonaceae but including at which a branch collapsed was recorded as the Amborellaceae, Austrobaileyaceae, Chloranthace- decay index. The larger the number, the more ae, and Illiciales), and Ranunculales (excluding robust a branch. A decay index of 1 indicates that Coriariaceae,Sargentodoxaceae, and Sabiaceae but the branch is present in all maximum parsimony including Eupteleaceae, Fumariaceae, and Papav- trees but collapses at maximum parsimony plus eraceae). (We will use these ordinal circumscrip- one step. tions throughout the rest of this paper; i.e., under Search D. There has been great interest in Piperales we will include Aristolochiaceae and Lac- recent cladistic studies to determine the basalmost toridaceae, unless stated otherwise.) The four mag- lineage of angiosperms. The Nymphaeales are often noliid lineages with monosulcate pollen types, (i) claimed as a sister group to all other angiosperms Magnoliales, (ii) Laurales, (iii) Piperales, and (iv) (Donoghue & Doyle, 1989; Hamby & Zimmer, Nymphaeales, plus the monocots (with the same 1992), but our results did not place them in such type of pollen), form a lade. The Illiciales, which a position. To evaluate the hypothesis of Nym- have anomalous tricolpate pollen (see below), also phaeles being the basalmost angiosperms, we per- fall into this monosulcate lade. The Piperales are formed a topology constraint experiment using the sister to all other monosulcates; Magnoliales/Nym- same set of taxa as in Search B, but with the phaeales, Laurales, and monocots form an unre- Nymphaeales constrained to be sister to all other solved trichotomy in the strict consensus tree. The angiosperms (using the CONSTRAINTS option of Ranunculales, Nelumbonaceae, and Sabiaceae, all PAUP). The search was conducted under the Fitch of which have tricolpate pollen, together with other criterion using 2,000 random sequence additions angiosperms possessing the same pollen type, form in the same manner as described in Search B. This a monophyletic group. This large lade corresponds is a more accurate means to determine the loss of to Walker & Doyle's (1975) nonmagnoliid dicots parsimony associated with an alternate topology; or Doyle & Hotton's (1991) "eudicots." The Ra- merely constraining the topology based on the nunculales are sister to the rest of the eudicots. In shortest trees found overall without performing another basal lineage of eudicots are Nelumbona- branch swapping is likely to result in an inaccurate ceae and Sabiaceae (with Platanaceae and Protea- assessment of tree length because it does not permit ceae; hamamelid I, Fig. 1). The Coriariaceae and character transformations to be optimized on dif- Sargentodoxaceae are not affiliated with the Mag- ferent branches than in the unconstrained trees. noliidae, the former being sister to the lade of Cucurbitaceae/Begoniaceae/Datiscaceae and the latter imbedded in the Fabaceae (both in rosid I, Fig. 1; also see fig. lIB in Chase et al., 1993). RESULTS Search B. A single island of 46 trees at a Search A. A strict consensus tree was com- length of 3,414 steps with C.I. of 0.275 (unique puted from 3,900 equally parsimonious trees at a substitutionsexcluded) and R.I. of 0.566 was found. length of 16,305 steps (Chase et al., 1993). These One of these maximum parsimony trees favored trees have a consistency index (C.I.; for potentially by the weighting criterion (Albert et al., 1993) is synapomorphous characters) of 0.102 and a re- shown with Fitch branch lengths optimized on it tention index (R.I.) of 0.638; a summary of this (ACCTRAN optimization;Fig. 2). Even though far tree is shown in Figure 1 (for the detailed version fewer taxa (82 species) were used in Search B, the of the strict consensus tree, see Chase et al., 1993). same general topology as Search A was obtained. Exclusive of Ceratophyllum, which is sister to all Search C. Two islands of 89 and 3 trees other flowering plants, the angiosperms are split respectively, at a length of 2,674 steps with C.I. into two clades that correspond to the two general of 0.346 (unique substitutions excluded) and R.I. pollen types, monosulcate and tricolpate (for the of 0.531 were found. For illustration, one tree from sake of convenience, we refer to angiosperms with each island was selected using the weighting cri- monosulcate and monosulcate-derived pollen types terion of Albert et al. (1993). The trees in the 542 Annals of the Missouri Botanical Garden

using relative rate tests (Gaut et al., 1992) dem- related taxa, and, like the effects of ancient hy- onstrated five-fold variation for rbcL, which still bridization, those of ancient polymorphisms would falls within the range cited above. Although 5-10- likely be minor relative to subsequent genetic di- fold differences in rates would appear potentially vergence. If lineages that contain polymorphisms to contribute enormous branch-length differences, diverged in a closely spaced manner and uneven the small overall rates involved mean that great sorting did take place, it is unlikely that any evi- divergence times would be a more important factor. dence of such variation within a progenitor could In addition, differences in rates appear to be highly be identified as such over the great amounts of lineage-correlated(for example, in graminoids;Gaut time involved in this study. Furthermore, poly- et al., 1992) rather than random, and if more morphisms are short-lived and are undocumented extensive sampling at lower taxonomic levels is for conservative, single-copy loci, such as rbcL. possible in these "fast" clades, then effects of rate At most these effects would be highly localized differences can be offset. among groups of closely related terminal taxa and, Most if not all rbcL rates will probably exist with adequate taxon sampling, would not be ex- within a relatively narrow "window," perhaps ap- pected to perturb greatly the results. proximating the range illustrated above (10-1lO Lateral transfers not involving exchange of ga- 10-11, V. A. Albert, M. W. Chase & J. F. Wendel, metes (by unknown mechanisms) may have oc- unpublished). If that is correct, lineage-specific rate curred between major lineages in the past (i.e., of inequalities are unlikely to be a primary factor in rbcL from a purple bacterium to a red algal an- branch attraction; rather, asymmetrical divergence cestor; Morden et al., 1992). Such transfers pres- times would be implicated because the product of ently appear rare among land plants and seed plants rate and time is the central parameter in consid- in particular; furthermore, artificial transforma- erations of potential systematic errors (see Albert tions are relatively difficult and often have a de- et al., 1993, this issue). Thus uneven sampling or stabilizingor transient effect on transformed plants. extinction of lineages may present greater problems While we must admit that this is an unknown area than do differences in rates. that could have played a role in certain anomalous placements in the rbcL trees (e.g., Montinia and Vahlia of Saxifragaceae sensu lato among asterids; EFFECTS OF LATERALGENOME TRANSFER, ANCESTRAL Fig. 15), at the same time we are POLYMORPHISMS,AND DIFFERENT not prepared to advocate it as a scenario MODES OF INHERITANCE until trees based on other data demonstrate a pattern consistent with such The trees presented here represent only infor- hypotheses (as in the example of Morden et al., mation from a single gene, and factors peculiar to 1992). Using evidence from studies of secondary its evolution could lead to erroneous results. Several chemistry and development, Morgan & Soltis of these phenomena are discussed below, but we (1993, this issue) build strong cases for the highly feel that their impact is likely minimal. Genome dispersed groupings found in their study of Saxi- transfers would result in all or parts of a genome fragaceae sensu lato. Furthermore, many of their being phylogenetically coherent (transferred as a findings were also congruent with recent investi- unit) at the time transit occurs (Doyle, 1992; Riese- gations of non-molecular characters. Rather than berg & Soltis, 1991), but before and after move- resorting to explanations involving lateral transfers ment most characters within genomes evolve in- of chloroplasts by mechanisms about which we can dependently (although still linked if on the same only speculate, we would prefer first to examine chromosome) and ought to be expected to contain specific cases from a cladistic perspective rather historical evidence. Hybridization is unlikely to in- than from that of current taxonomic schemes. fluence phylogenetic analyses except at lower tax- An rbcL tree is not solely a maternal tree. Al- onomic levels. Even matings between divergent though chloroplast transmission is principally ma- parents still occur within portions of families (and ternal and uniparental, several groups exhibit a usually among closely related species in a genus) paternal (e.g., conifers) or biparental pattern (Neale rather than between families. Parental taxa at the et al., 1986; Szmidt et al., 1987; Whatley, 1982; time of an ancient hybridization also were likely Wagner et al., 1987; Corriveau & Coleman, 1988; closely related, and in these genomes highly con- White, 1990; Owens & Morris, 1991). Others that served loci, such as rbcL, would have been similar have been thought most probably maternal, such or even identical. as Liriodendron and Magnolia (Corriveau & Cole- Ancestral polymorphisms (Pamilo & Nei, 1988; man, 1988), consistently exhibit 5-15% paternal Wu, 1991; Doyle, 1992) also affect only closely inheritance (Sewell et al., 1993). The potential Volume 80, Number 3 Chase et al. 543 1993 Phylogenetics of Seed Plants

effects of a mixed pattern of inheritance on gene An additional implication of adding more taxa trees are also unlikely to affect studies at interfam- is that the results presented here may not be stable ilial levels and above. or reflective of those of an analysis of two, three, or four times as many sequences. We point to two regions of the general topology that are morpho- GENES VERSUS TAXA logically quite heterogeneous and that we believe One of the more persistent controversies sur- are possibly generated by undersampling: hama- rounding molecular phylogenetic studies has cen- melid II (Figs. 2A, B, 7A, B) and rosid I (Figs. tered on whether results, at a given taxonomic 2A, B, 1 A, B, C). In the former, the critical taxa level, will be "improved" more by adding additional needed to provide more appropriate relationships taxa sequenced for the same gene or by adding could be some of the still-unsampled families, but sequence analyses of additional genes for the same these groups (represented by Lambertia, Nelumbo, set of taxa (analyzed simultaneously or each per- Platanus, and Sabia) could just as likely represent formed independently for assessments of congru- the isolated relicts of now largely extinct lineages. ence; Pamilo & Nei, 1988; Wu, 1991). From the In the case of rosid I, only a small percentage of standpoint of corroboration, phylogenetic studies the families has been sampled (for instance, only of other data sets are absolutely crucial. Never- 7 of 24 families in the Violales: Dilleniidae), and theless, it seems quite clear from our work on these it is in such a case that we might predict "curious" data sets that ideas of relationships have changed sister-group relationships. Topological instability considerably as more taxa have been added. We often occurs in cases where long terminal branches suspect that no single gene sequence can provide are next to short internal branches, that is, hy- reasonablehypotheses of relationshipsof seed plants pothesized relationships are drastically altered by when sampled uncritically and superficially, but the addition of related taxa that "break up" long perhaps erroneous results from one locus would be branches. Two surprising pairs of sister taxa from "corrected" by stronger signals present in the oth- Search I, Erythroxylum-Viola and Ochna-Dry- ers. Data to evaluate this most critical question do petes, are quite divergent and connected to each not exist: would separately analyzing 20 gene se- other by relatively short branches (Fig. 1 IA; branch quences for the same set of 25 to 30 taxa produce lengths shown only in 11 B). In Search II, the well supported relationships? addition of Reinwardtia displaced Erythroxylum We are convinced that adding representatives from Viola to Drypetes, leaving Ochna to stand of the still numerous and diverse families absent isolated from any other taxon (Fig. 1I B). In such from this analysis has the potential to enhance situations, assessments of relationships are difficult, assessments of relationships at all levels (despite but, as more closely related species are added, obvious computational complications). A great deal distinguishing synapomorphies for families and of the variation present within the gene still remains groups of families as distinct from autapomorphies to be sampled taxonomically. Studies in several for individual species will become more reliable. groups of plants indicate that rbcL often can be We would not argue that the relationships found valuable at rather low taxonomic levels (Albert, in for rosid I are "better" in Search I than in Search press, in the slipper orchids; S. Graham, B. Morton II (or vice versa), but rather point these out as & S. Barrett, unpublished, in Eichhornia; R. Price areas of the trees that require additional sampling & J. Palmer, unpublished, in Pelargonium; S. and in which many of the relationships suggested Williams & M. Chase, submitted, in Drosera; Xi- by these analyses of rbcL have little or no mor- ang et al., 1993, this issue, in Cornus). What can phological support. be concluded from these studies is that although it is absolutely critical that more genes be studied VALUE OF THE BROAD ANALYSIS to provide corroboration, it is unlikely that any of these studies will make a contribution to under- We view the relatively robust internal support standing seed-plant relationships if the level of sam- found by evaluations of portions of the broad anal- pling is too sparse. Only empirical studies will ul- ysis as an indication that rbcL sequences contain timately resolve the ""genes versus taxa" information relevant to the evolutionary history of controversy. We estimate that by the time this angiosperms (Fig. 16, on Hamamelidae and most paper is in print, more than 1,200 sequences of of the other papers in this issue; in particular see rbcL from seed plants will exist, and future studies Conti et al., 1993, and Rodman et al., 1993, for will undoubtedly benefit from this enormous da- which comparable cladistic analyses of non-molec- tabase. ular data are also available and compared). Well- 544 Annals of the Missouri Botanical Garden

characterized families and groups of families (as treated these groups in a manner somewhat similar evidenced in several more restricted studies of mor- to our topology); (iii) relationships of Nepenthaceae phology, anatomy, and secondary chemistry; e.g., and Droseraceae to Caryophyllidae sensu lato (Fig. Rodman, 1991a, b; Hufford, 1992) are largely 9A, B); and (iv) specific associations of numerous congruent with our results. Other evidence sup- problematicgenera like Dillenia (among rosids near ports the monophyly of the angiosperms (Doyle & Caryophyllidae, Fig. 9A, or near the base of the Donoghue, 1986, 1992), monocots (Dahlgren et asterids, Fig. 12B; see Olmstead et al., 1993, this al., 1985), and eudicots (Donoghue & Doyle, 1989). issue), Impatiens (near members of Ebenales and Likewise, the position of Ceratophyllum as sister Ericales; Fig. 13A, B), and Nelumbo and Lam- to the rest of the angiosperms compares favorably bertia (among lower hamamelids; Fig. 7A, B). with the fossil record (Les, 1988; fossil fruits from Placement of most of these genera and families 120 million years ago are identical to extant fruits, has varied substantially among recently proposed D. Dilcher, pers. comm.). The status of the families classifications (although no one has suggested the of the Papaverales-Ranunculales collectively as relationships found here), and their positions in this sister to the rest of the eudicots (Figs. 1, 2) also study will undoubtedly add to the controversies. finds ample external support (Donoghue & Doyle, Because additional lineages were present in 1989, among others). Search II and different methods were used to con- The results reported here do find some "sur- struct the trees, it is impossible to evaluate whether prising" relationships. Several families, such as the topology found in Search II represents a dif- Chenopodiaceae (Fig. 9A, B) and Berberidaceae ferent island of trees. Indeed, multiple islands of with only two representatives each in our studies, equally parsimonious trees were found in other are paraphyletic to other families. These results do studies in this issue: Morgan & Soltis (1993), Olm- not surprise us because sampling has been dem- stead et al. (1993), and Qiu et al. (1993). Certainly onstrated repeatedly to be a major factor, and shifts of some taxa, especially Paeonia from a basal insufficient or uneven sampling can generate anom- asterid to sister of Crassulaceae and Saxifragaceae alous relationships. Concomitantly, our results em- sensu stricto, suggest a radically different expla- phasize the need for studies of familial limits to nation of the distribution of at least some char- include much better taxon sampling than is gen- acters. Paeonia is well supported internally in its erally the case in these two analyses. Some of these new position (its sister status to Ribes does not instances of paraphyly may be accurate; many decay even at five steps less parsimonious; Fig. pairs of temperate, herbaceous/woody, tropical 16). Shifts of Dillenia, Gunnera, Santalales, and families have long been suspected of being unnat- Vitis seem, at first glance, to be major alterations ural (for example, Lamiaceae are derived within of position, but branches are so short near the split Verbenaceae and Brassicaceae within Capparaceae between asterids and rosids that these could not in our studies). involve many additional steps in either topology Other "major" findings of this study, although (Fig. 2B). discordant when viewed from the perspective of Although the trees of Search II are preferred various taxonomic treatments, find support from to those of Search I because some of them were recent studies of non-molecular characters. For swapped on to completion and therefore are more example, the placement of Ericales as sister to likely to represent at least a local optimum, tax- asterids (Figs. 1, 2) was also found by Hufford onomic conclusions based on either search are un- (1992), and the monophyly of most mustard-oil timely. When faced with the fact that large num- families (Fig. 1 A, B) was previously suggested by bers of angiosperm families are still unrepresented Rodman (1991 b). Other sets of relationships are in the rbcL data set, we would argue that a valid unique to this analysis and require more thorough assessment of the most appropriate positions of morphological and molecular studies. These in- many taxa, such as Dillenia, Gunnera, and Vitis, clude: (i) the position of several families with poly- grossly premature. Some conclusions, polyphyly of petalous corollas and supposed affinities to Saxi- Hamamelidae and Dilleniidae, for example, seem fragaceae among each major lineage of Asteridae well supported now. (requiring a hypothesized reversal of the sympet- This study is noteworthy not only for its scope alous condition in Escallonia, Montinia, Phyllono- but also for the large number of contributors whose ma, and Vahlia (all Saxifragaceae sensu lato); (ii) unpublished sequences made up the bulk of the nesting of dilleniid orders Capparales, Malvales, data analyzed. This wide collaboration was advan- Theales, and Violales among rosid clades (the sys- tageous to all workers; many found that taxa se- tems of both Dahlgren, 1980, and Thorne, 1992, quenced by other laboratories supposedly working Volume 80, Number 3 Chase et al. 545 1993 Phylogenetics of Seed Plants

on distantly related groups (in recent taxonomic these analyses represent an attempt to improve schemes) fell into or near their group of interest. both our understanding of seed-plant evolution and Prime examples of this are the close phylogenetic methods of phylogenetic inference. Corroboration relationship of Nepenthaceae and Droseraceae to by other data sets analyzed in a similar fashion is families of Caryophyllidae, Pittosporaceae to Api- by far the most significant measure of relationships aceae-Araliaceae, Malvales and Capparales (both proposed here, and we hope this process of eval- "dilleniids") to Sapindales, and Corokia (Corna- uation will be innervated by our efforts. ceae) to Asteraceae. Broad analyses are thus important in providing LITERATURE CITED evaluations of a priori assumptions about appro- ALBERT, V. A. Phylogeny of the slipper orchids (Cypri- priate sets of study taxa for more focused and pedioideae: Orchidaceae) from congruent morpho- and molecular data sets. Nordic J. Bot. (in studies; they should be formalized so that logical rigorous press). someone takes the initiative to perform them. Gov- & B. D. MISHLER. 1992. On the rationale and ernmental funding agencies should facilitate stud- utility of weighting nucleotide sequence data. Cladis- ies, such as the one presented here, that are well tics 8: 73-83. beyond the scope of individual laboratories. This , M. W. CHASE & B. D. MISHLER. 1993. Char- acter-state weighting for cladistic analysis of protein- study demonstrates the potential of this kind of coding DNA sequences. Ann. Missouri Bot. Gard. analysis, but no single individualor laboratory could 80: 752-766. have received formal support for sampling this , B. D. MISHLER & M. W. CHASE. 1992a. Char- diverse set of taxa and performing the phylogenetic acter-state weighting for restriction site data in phy- logenetic reconstruction, with an example from chlo- analysis; it would have been deemed by reviewers roplast DNA. Pp. 369-403 in P. S. Soltis, D. E. too broad and too unfocused. Although extramural Soltis & J. J. Doyle (editors), Molecular Systematics funds supported most of the other studies in this in Plants. Chapman & Hall, New York. issue, no support was received specifically for the , S. E. WILLIAMS & M. W. CHASE. 1992b. broad analysis. It is the investment in individual Carnivorous plants: Phylogeny and structural evo- lution. Science 257: 1491-1495. to sup- studies that justifies a further expenditure ARCHIE, J. W. 1989. A randomization test for phylo- port syntheses that supply an essential overall per- genetic information in systematic data. Syst. Zool. spective, even though they may be necessarily 38: 219-252. approximate. BoUSQUET, J., S. H. STRAUSS, A. D. DOERKSEN & R. A. PRICE. 1992. Extensive variation in evolutionary The benefits of performing this largest-yet phy- rate of rbcL gene sequences among seed plants. Proc. logenetic study of seed plants lie not only in support Natl. Acad. 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We have performed 685. none of the essential experiments that these results CORRIVEAU, J. L. & A. W. COLEMAN. 1988. Rapid suggest (topological constraints, removal of char- screening method to detect potential biparental in- heritance of plastid DNA and results for over 200 acters, combining morphological with molecular angiosperm species. Amer. J. Bot. 75: 1443-1458. data, etc.) and have not developed implications CRANE, P. R. 1985. Phylogenetic analysis of seed plants these topologies may have for specific character and the origin of the angiosperms. Ann. Missouri Bot. transformations in seed plants or molecular evo- Gard. 72: 716-793. clades and in the lution of rbcL or RuBisCO. Believing that serious 1988. Major relationships "higher" gymnosperms. Pp. 218-272 in C. B. Beck consideration of the significance of these general (editor), Origin and Evolution of Gymnosperms. Co- topologies is best handled at the more manifest lumbia Univ. 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DAHLGREN, R. T. 1980. A revised system of classifi- approach using the bootstrap. Evolution 39: 783- cation of the angiosperms. Bot. J. Linn. Soc. 80: 791. 91-124. . 1990. PHYLIP manual, version 3.3. Univ. & K. BREMER. 1985. Major clades of the of California, University Herbarium, Berkeley, Cal- angiosperms. Cladistics 1: 349-368. ifornia. , H. T. CLIFFORD & P. F. YEO. 1985. The FITCH, W. M. 1971. Toward defining the course of Families of the Monocotyledons: Structure, Evolu- evolution: Minimum change for a specific tree to- tion, and Taxonomy. Springer-Verlag, New York. pology. Syst. Zool. 20: 406-416. DOEBLEY, J., M. DURBIN, E. M. GOLENBERG, M. T. CLEGG GAUT, B. S., S. V. MUSE, W. D. CLARK & M. T. CLEGG. & D. P. MA. 1990. Evolutionary analysis of the 1992. Relative rates of nucleotide substitution at large subunit of carboxylase (rbcL) nucleotide se- the rbcL locus of monocotyledonous plants. J. Molec. quence among the grasses (Gramineae). Evolution Evol. 35: 292-303. 44: 1097-1108. GIANNASI, D. E., G. ZURAWSKI, G. LEARN & M. T. CLEGG. DONOGHUE, M. J. & J. A. DOYLE. 1989. Phylogenetic 1992. Evolutionary relationships of the Caryophyl- studies of seed plants and angiosperms based on lidae based on comparative rbcL sequences. Syst. morphological characters. Pp. 18 1-193 in B. Fern- Bot. 17: 1-15. holm, K. Bremer & H. J6rnvall (editors), The Hi- GOLDBLATT, P., P. C. HOCH & L. M. MCCOOK. 1992. erarchy of Life: Molecules and Morphology in Phy- Documenting scientific data: The need for voucher logenetic Analysis. Elsevier Science Publishers, specimens. Ann. Missouri Bot. Gard. 79: 969-970. Amsterdam. HAMBY, R. K. & E. A. ZIMMER. 1992. Ribosomal RNA , R. G. OLMSTEAD, J. F. SMITH & J. D. PALMER. as a phylogenetic tool in plant systematics. Pp. 50- 1992. Phylogenetic relationshipsof Dipsacales based 91 in P. S. Soltis, D. E. Soltis & J. J. Doyle (editors), on rbcL sequences. Ann. Missouri Bot. Gard. 79: Molecular Systematics in Plants. Chapman & Hall, 333-345. New York. , J. A. DOYLE, J. GAUTHIER, A. G. KLUGE & T. HARRIS, P. J. & R. D. HARTLEY. 1980. Phenolic con- ROWE. 1989. The importance of fossils in phylog- stituents of cell walls of monocotyledons. Biochem. eny reconstruction. Ann. Rev. Ecol. Syst. 20: 431- Syst. Ecol. 8: 153-160. 460. HENDY, M. D. & D. PENNY. 1989. A framework for DOWNIE, S. R. & J. D. PALMER. 1992. Use of chlo- the quantitative study of evolutionary trees. Syst. roplast DNA rearrangements in reconstructing plant Zool. 38: 297-309. phylogeny. Pp. 14-35 in P. S. Soltis, D. E. Soltis HUFFORD, L. 1992. Rosidae and their relationships to & J. J. Doyle (editors), Molecular Systematics in other nonmagnoliid dicotyledons: A phylogenetic Plants. Chapman & Hall, New York. analysis using morphologicaland chemical data. Ann. DOYLE, J. A. & M. J. DONOGHUE. 1986. Seed plant Missouri Bot. Gard. 79: 218-248. phylogeny and the origin of the angiosperms: An JANSEN, R. K. & J. D. PALMER. 1988. Phylogenetic experimental cladistic approach. Bot. Rev. 52: 321- implications of chloroplast DNA restriction site vari- 431. ation in the Mutisieae (Asteraceae). Amer. J. Bot. & - 1992. Fossils and seed plant phy- 75: 751-764. logeny reanalyzed. Brittonia 44: 89-106. KXLLERSJ6, M., J. S. FARRIS, A. G. KLUGE & C. BULT. & L. J. HICKEY. 1976. Pollen and leaves from 1992. Skewness and permutation.Cladistics 8: 275- the mid-CretaceousPotomac Group and their bearing 287. on early angiosperm evolution. Pp. 139-206 in C. KIM, K.-J., R. K. JANSEN, R. S. WALLACE, H. J. MICHAELS B. Beck (editor), Origin and Early Evolution of An- & J. D. PALMER. 1992. Phylogenetic implications giosperms. Columbia Univ. Press, New York. of rbcL sequence variation in the Asteraceae. Ann. DOYLE, J. J. 1992. Gene trees and species trees: Mo- Missouri Bot. Gard. 79: 428-445. lecular systematics as one-character taxonomy. Syst. KIMURA, M. 1980. A simple method for estimating Bot. 17: 144-163. evolutionary rate of base substitutions through com- & HOLTON. 1991. Diversification of early an- parative studies of nucleotide sequences. J. Molec. giosperm pollen in a cladistic context. Pp. 169-195 Evol. 16: 111-120. in S. Blackmore & S. H. Barnes (editors), Pollen and . 1981. Estimation of evolutionary distances Spores. Clarendon Press, Oxford. between homologousnucleotide sequences. Proc. Natl. DUVALL, M. R., M. T. CLEGG, M. W. CHASE, W. D. Acad. U.S.A. 78: 454-458. CLARK, W. J. KRESS, H. G. HILLS, L. E. EGUIARTE, KRON, K. A. & M. W. CHASE. 1993. Systematics of J. F. SMITH, B. S. GAUT, E. A. ZIMMER & G. H. the Ericaceae, Empetraceae, Epacridaceae and re- LEARN. 1993. Phylogenetic hypotheses for the lated taxa based upon rbcL sequence data. Ann. monocotyledonsconstructed from rbcL sequence data. Missouri Bot. Gard. 80: 735-741. Ann. Missouri Bot. Gard. 80: 607-619. LES, D. H. 1988. The origin and affinities of the Cer- FAITH, D. & P. CRANSTON. 1991. Could a cladogram atophyllaceae. Taxon 37: 326-345. this short have arisen by chance alone? Cladistics 7: 9 D. K. GARVIN & C. F. WIMPEE. 1991. Mo- 1-28. lecular evolutionary history of ancient aquatic an- FELSENSTEIN, J. 1978. Cases in which parsimony or giosperms. Proc. Natl. Acad. U.S.A. 88: 10119- compatibility methods will be positively misleading. 10123. Syst. Zool. 27: 401-410. LOCONTE, H. & D. W. STEVENSON. 1991. Cladistics of 1981. Evolutionary trees from DNA sequenc- the Magnoliidae. Cladistics 7: 267-296. es: A maximum likelihood approach. J. Molec. Evol. MADDISON, D. R. 1991. Discovery and importance of 17: 368-376. multipleislands of most-parsimonioustrees. Syst. Zool. 1985. Confidence limits on phylogenies: An 40: 315-328. Volume 80, Number 3 Chase et al. 547 1993 Phylogenetics of Seed Plants

MARTIN, P. G. & J. M. DOWD. 1991. Studies of an- consequences of cytoplasmic gene flow in plants. giosperm phylogenies using protein sequences. Ann. Evol. Trends P1. 5: 65-84. Missouri Bot. Gard. 78: 296-337. RITLAND,K. & M. T. CLEGG. 1987. Evolutionary anal- MICHAELS, H. J., K. M. SCOTT, R. G. OLMSTEAD, T. SZARO, yses of plant DNA sequences. Amer. Naturalist 130: R. K. JANSEN & J. D. PALMER. 1993. Interfamilial S74-S100. relationships of the Asteraceae: Insights from rbcL RODMAN, J. 1991a. A taxonomic analysis of glucosi- sequence variation. Ann. Missouri Bot. Gard. 80: nolate-producingplants, Part 1: Phenetics. Syst. Bot. 742-765. 16: 598-618. MORDEN, C. W., C. F. DELWICHE, M. KUHSEL & J. D. 1991b. A taxonomic analysis of glucosinolate- PALMER. 1992. Gene phylogenies and the endo- producing plants, Part 2: Cladistics. Syst. Bot. 16: symbiotic origin of plastids. Biosystems 28: 75-90. 619-629. MORGAN, D. R. & D. E. SOLTIS. 1993. Phylogenetic , R. PRICE, K. KAROL, E. CONTI, K. SYTSMA & relationshipsamong members of Saxifragaceae sensu J. D. PALMER. 1993. Nucleotide sequences of the lato based on rbcL sequence data. Ann. Missouri Bot. rbcL gene indicate monophyly of mustard oil plants. Gard. 80: 631-660. Ann. Missouri Bot. Gard. 80: 686-699. NEALE, D. B., N. C. WHEELER & R. W. ALLARD. 1986. SCHWARZWALDER, R. & D. L. DILCHER. 1991. System- Paternal inheritance of chloroplast DNA in Douglas- atic placement of the Platanaceae in the Hamamel- fir. Canad. J. Forest Res. 16: 1152-1154. idae. Ann Missouri Bot. Gard. 78: 962-969. NIXON, K. C. & G. I. DAVIS. 1991. Polymorphic taxa, SEWELL, M., M. W. CHASE, Y.-L. Qiu & C. R. PARKS. missing values, and cladistic analyses. Cladistics 7: 1993. Trace paternalinheritance of chloroplastDNA 233-241. in Liriodendron and Magnolia (Magnoliaceae).Amer. OLMSTEAD, R. G., H. J. MICHAELS, K. M. SCOTT& J. D. J. Bot. 80 (in press). PALMER. 1992. Monophyly of the Asteridae and SMITH, J. F., W. J. KRESS & E. A. ZIMMER. 1993. identification of their major lineages inferred from Phylogenetic analysis of the Zingiberales based on DNA sequences of rbcL. Ann. Missouri Bot. Gard. rbcL sequences. Ann. Missouri Bot. Gard. 80: 620- 79: 249-265. 630. , B. BREMER, K. M. SCOTT& J. D. PALMER. SOLTIS, D. E., P. S. SOLTIS, M. T. CLEGG& M. DUR- 1993. A parsimony analysis of the Asteridae sensu BIN. 1990. rbcL sequence divergence and phylo- lato based on rbcL sequences. Ann. Missouri Bot. genetic relationships in Saxifragaceae sensu lato. Gard. 80: 700-722. Proc. Natl. Acad. U.S.A. 87: 4640-4644. OWENS, J. N. & S. J. MORRIS. 1991. Cytological basis SWOFFORD,D. L. 1991. PAUP: Phylogenetic Analysis for cytoplasmic inheritance in Pseudotsuga menzie- Using Parsimony, Version 3.0r. Computer program sii. II. Fertilization and proembryo development. distributed by the Illinois Natural History Survey, Amer. J. Bot. 78: 1515-1527. Champaign, Illinois. PALMER, J. D., R. K. JANSEN, H. J. MICHAELS, M. W. & G. J. OLSEN. 1990. Phylogeny reconstruc- CHASE & J. M. MANHART. 1988. Analysis of chlo- tion. Pp. 411-501 in D. M. Hillis & C. Moritz roplast DNA variation. Ann. Missouri Bot. Gard. 75: (editors), Molecular Systematics. Sinauer Associates, 1180-1206. Sunderland, Massachusetts. PAMILO, P. & M. NEI. 1988. Relationships between SZMIDT, A. E., T. ALDtN & J.-E. HALLGREN. 1987. gene trees and species trees. Molec. Biol. Evol. 5: Paternal inheritance of chloroplast DNA in Larix. 568-583. P1. Molec. Biol. 9: 59-64. PENNY, D., M. D. HENDY & M. A. STEEL. 1992. Prog- TAKHTAJAN,A. 1980. Outline of the classification of ress with methods for constructing evolutionary trees. floweringplants (Magnoliophyta).Bot. Rev. 46: 225- Trends Ecol. Evol. Biol. 7: 73-79. 359. 1987. Systema DE PINNA, M. C. C. 1991. Concepts and tests of ho- Magnoliophytorum. Nauka, mology in the cladistic paradigm. Cladistics 7: 367- Leningrad. [In Russian.] 394. TAYLOR, D. W. & L. J. HICKEY. 1992. Phylogenetic evidence for the herbaceous origin of angiosperms. PLATNICK, N. I., C. E. GRISWOLD & J. A. CODDINGTON. P1. Syst. Evol. 180: 137-156. 1991. On missing entries in cladistic analyses. Cla- THORNE, R. F. new in distics 7: 337-343. 1983. Proposed realignments the angiosperms. Nordic J. Bot. 3: 85-117. PRICE, R. A. & J. D. PALMER. 1993. Phylogenetic 1992. An updated classification of the flow- relationships of the Geraniaceae and Geraniales from ering plants. Aliso 13: 365-389. rbcL sequence comparisons.Ann. MissouriBot. Gard. WAGNER, D. B., G. R. M. A. 80: 661-671. FURNIER, SAGHAI-MAROOF, S. M. WILLIAMS,D. B. DANCIK & R. W. ALLARD. QIu, Y.-L., M. W. CHASE, D. H. LES & C. R. PARKS. 1987. ChloroplastDNA polymorphismsin lodgepole 1993. Molecular phylogenetics of the Magnoliidae: and jack pines and their hybrids. Proc. Natl. Acad. Cladisticanalyses of nucleotide sequences of the plas- U.S.A. 84: 2097-2100. tid gene rbcL. Ann. Missouri Bot. Gard. 80: 587- WENDEL, J. F. & V. A. ALBERT. 1992. Phylogenetics 606. of the cotton genus (Gossypium): Character-state RAUBESON, L. A. & R. K. JANSEN. 1992. Chloroplast weighted parsimony analysis of chloroplast-DNA re- DNA evidence on the ancient evolutionary split in striction site data and its systematic and biogeograph- vascular land plants. Science 255: 1697-1699. ic implications. Syst. Bot. 17: 115-143. RETTIG, J. H., H. D. WILSON & J. M. MANHART. 1992. WHATLEY,J. M. 1982. Ultrastructure of plastid inher- Phylogeny of the Caryophyllales-Gene sequence itance: Green algae to angiosperms. Bot. Rev. 57: data. Taxon 41: 201-209. 527-569. RIESEBERG, L. H. & D. E. SOLTIS. 1991. Phylogenetic WHITE, E. E. 1990. Chloroplast DNA in Pinus mon- 548 Annals of the Missouri Botanical Garden

ticola. 2. Survey of within-species variability and Currentaddress for LEE: Centro de Ecologia, Universidad detection of heteroplasmic individuals.Theoret. Appl. Nacional Auton6ma de Mexico, Apartado Postal 70-275, Gen. 79: 251-255. C.U., C.P. 04510, Mexico, D.F., Mexico. Current address WILSON, M. A., B. GAUT & M. T. CLEGG. 1990. Chlo- for BSG: Department of Statistics, North Carolina State roplast DNA evolves slowly in the palm family. Molec. University, Raleigh, North Carolina 27695, U.S.A. Cur- Biol. Evol. 7: 303-314. rent address for MRD: Office of Biotechnology, Iowa State YOUNG, D. J. & L. WATSON. 1970. The classification lJniversity, Ames, Iowa 50011-3260, U.S.A. of dicotyledons: A study of the upper levels of hi- 8 Department of Biology, Indiana University, Bloo- erarchy. Austral. J. Bot. 18: 387-433. mington, Indiana 47405, U.S.A. Currentaddress for RAP: Wu, C.-L. 1991. Inference of species phylogeny in Department of Botany, University of Georgia, Athens, relation to segregation of ancient polymorphisms. Georgia 30602, U.S.A. Genetics 127: 429-435. 9 Department of Biology, Texas A & M University, XIANG, Q.-Y., D. E. SOLTIS,D. R. MORGAN& P. S. SOLTIS. College Station, Texas 77843, U.S.A. Current Address 1993. Phylogenetic relationshipsof Cornus L. sensu for JHR: Department of Biology, College of the Ozarks, lato and putative relatives inferred from rbcL se- Point Lookout, Missouri 65726, U.S.A. quence data. Ann. Missouri Bot. Gard. 80: 723- 10Department of Botany, University of Wisconsin, 734. Madison, Wisconsin 53706, U.S.A. ZURAWSKI, G. & M. T. CLEGG. 1987. Evolution of 11Department of Biological Sciences, Bowling Green higher-plant chloroplast DNA-coded genes: Implica- State University, Bowling Green, Ohio 43403, U.S.A. tions for structure-function and phylogenetic studies. 12 Department of Botany, Smithsonian Institution, Ann. Rev. PI. Phys. 38: 391-418. Washington, D.C. 20560, U.S.A. Current address for JFS: Department of Biology, Boise State University, Boise, Idaho 83725, U.S.A. 13 Department of Botany, Arizona State University, Tempe, Arizona 85287, U.S.A. 2 Department of Biology, University of North Carolina, 14 Institutionen F6r Systematisk Botanik, Uppsala Chapel Hill, North Carolina 27599-3280, U.S.A. Current Universitet, Uppsala S-751 21, Sweden. address for MWC: Laboratory of Molecular Systematics, 15Department of Botany, University of Texas, Austin, Royal Botanic Gardens, Kew, Richmond, Surrey TW9 Texas 78713, U.S.A. 3AB, United Kingdom. Current address for KAK: De- 16 Department of Forest Resources and Plant Biology, partment of Biology, Wake Forest University, Winston- University of Minnesota, St. Paul, Minnesota 55108- Salem, North Carolina 27109, U.S.A. Current address 8112, U.S.A. for VAA: Institutionen F6r Systematisk Botanik, Uppsala 17 Department of Forest Science, FSL 020, Oregon Universitet, Uppsala S-751 21, Sweden. State University, Corvallis, Oregon 97331-7505, U.S.A. 3Department of Botany, Washington State University, 18 Department of Botany, University of Tennessee, Pullman, Washington 99164-4238, U.S.A. Knoxville, Tennessee 37996-1100, U.S.A. Current ad- 4Department of E. P. 0. Biology, University of Col- dress: Rancho Santa Ana Botanic Garden, Claremont, orado, Boulder, Colorado 80309-0334, U.S.A. California 91711-3101, U.S.A. 5 Department of Biological Sciences, University of Wis- 19 Department of Biology, Lebanon Valley College, consin, Milwaukee, Wisconsin 53201, U.S.A. Current Annville, Pennsylvania 17003-0501, U.S.A. address for DHL: Department of Ecology and Evolution- 20 School of Biological Sciences, University of New ary Biology, University of Connecticut, Storrs, Connect- South Wales, P.O. Box 1, Kensington, New South Wales, icut 06269-3042, U.S.A. Australia. 6 Department of Botany, Duke University, Durham, 21 Department of Biological Sciences, Wayne State Uni- North Carolina 27708-0338, U.S.A. Current address: versity, Detroit, Michigan 48202, U.S.A. Department of Integrative Biology, University of Califor- 22 Department of Botany, University of Toronto, To- nia, Berkeley, California 94720, U.S.A. ronto, Ontario M5S 3B2, Canada. 7 Department of Botany and Plant Sciences, University 23 Department of Biology, Boston University, Boston, of California, Riverside, California 92521-0124, U.S.A. Massachusetts 02215, U.S.A. 550 Annals of the Missouri Botanical Garden

lA

Asteridae -- including Ericales,Primulales, Ebenales,Santalales, Apiales,Comnales, and some Rosales

Rosidae -- including Violales, Malvales, mustard-oilfamilies, higherHamamelidae, andCaryophyllidae

\ \hamameis NNSSSS ~~~~~~~~~~ranunculicts paleoherbs

monocots

Laurales Magnoliales Ceratophyllaceae gnetophytes Pinaceae other conifers cycads FIGURE 1. Summaries of the major clades identified in: (A) the combinable component consensus tree of 500 equally parsimonious trees found for 475 taxa using the character-state weighting method of Albert et al. (1993, this issue); and (B) the strict consensus tree of 3,900 equally parsimonious trees for 499 taxa found using the Fitch (even weights) criterion. These are ingroup networks arranged arbitrarily with the cycads sister to all other seed plants. Volume 80, Number3 Chase et al. 551 1993 Phylogenetics of Seed Plants

1B

Asteridae-- including Ericales, Primulales, Ebenales, Santalales, Apiales, Cornales,and some Rosales

Rosidae -- including Violales, Malvales, mustard-oilfamilies, and higher Hamameiidae

caryophyllids Gunneraceae hamamelids ranunculids paleoherbsII Magnoliales Laurales

monocots

paleoherbsI Ceratophyllaceae gnetophytes other conifers Pinaceae Ginkgo cycads 552 Annals of the Missouri Botanical Garden

2A asterid I asterid II asterid III asterid IV asterid V rosid IV rosid III rosid II rosid I hamamelid II hamamelid I ranunculids paleoherbs monocots Laurales .Magnoliales Ceratophyllum Gnetales Pinaceae other conifers cycads

FIGURE 2. Summaries of the same topologies as in Figure 1. In B, Fitch branch lengths are optimized from a single tree; optimization on consensus trees is likely to overestimate branch lengths. Names of the specific clades identified do not conform to the composition of families used in most taxonomic schemes, but rather are designated with respect to the components of the major lineages (i.e., by the subclass name for the majority of taxa included, except for the heterogeneous hamamelid I, which is so designated because of its position and inclusion of Platanaceae). Names of each clade correspond to groups shown in Figures 3-15. Clades marked with a "?" in B are those that differ significantly in position or composition from A. Volume 80, Number 3 Chase et al. 553 1993 Phylogenetics of Seed Plants

2B 65 asterid I 3 4 asterid II 6 10 asterid III 4| 5 asterid IV 3 1 6 asterid V? 9 I rosid I 5 5 rosid II 5 > rosid III 5 L 9caryophyllids? Gunnera? 4 8 hamamelid II 18 = 5 hamamelid I ranunculids 4 2 paleoherb II ? 6 Magnoliales

591 | 11 | v monocots Laurales 39 1 12 paleoherb I? 44 Ceratophyllum 18 61 Gnetales - 16 other conifers

l | _ 32 Pinaceae 28 L 23 Ginkgo? cycads 554 Annals of the Missouri Botanical Garden

3A angiosperms Gnetum Gnetaceae Welwitschia Welwitschiaceae Ephedra Ephedraceae Abies Keteleeria r- Pseudotsuga Larix Picea sitchensis Picea pungens Pinaceae Pinus grtfflthii Pinus radiata Cedrus Pseudolarix Tsuga Callitris Cpesca Widdringtonia CuPressaceac Taxodium LMetasequoia Taxodiaceae Sequoiadendron Podocarpus Podocarpaceae Cycas Cycadacae Microcycast Zamia Chigua Bowenia 7 -EgastuIt { E~Macrozamiat Zamiaceae ast H ~~Encephalartos 11_ast V I Ceratozamiai rosIII ~Stangeria ros I 1ll Lepidozamiat~ I _ rsnMDioon . _Xmon~~~~pal _ ~~~mag mgnegcer [ ~ mcyc~~~~conJ FIGURE3. A portion of the overall analysis showing the "gymnosperms." (Numbers above the branches in B are the numbers of substitutions optimized onto one tree randomly selected from the 3,900 saved in Search II.) Note that A is the consensus tree of Search I, whereas B is a single tree with branches not present in the strict consensus of Search II marked by an arrow. Genera marked with a "t" in A were omitted from Search II; genera marked with an asterisk in B were not available for Search I. Volume80, Number3 Chase et al. 555 1993 Phylogenetics of Seed Plants

3B 59 angiosperms 39 Mag42 Gnetum Gnetaceae 61 52 Welwitschia Welwitschiaceae 70 Ephedra Ephedraceae 38 22 t Sciadopitys* Taxodiaceae 6 Taxus* Taxaceae Cephalotaxus* Cephalotaxaceae

d Widdringtonia Cupressaceae 7 X ~ Metasequoia Sequoiadedron lAt9hArotaxis* Taxodiaceae 18 30 Taxodium 65 Podocarpus Podocarpaceae 7 Abies 5 ~Keteleeria 4 Pseudolarix

8 Pseudotsuga l 1 Larix Pinaceae 32 ~~20 Picea 12 Pinus gniffithii Pinus radiata 28 12 Cedrus 23 Ginkgo* Ginkgoacae 31 Cycas Cycadacae 18 Bowenia

I -EntEncephalartos Zamiaceae st1V Stangeria 4A eudicots Euryalet Victoria INymphaeaceaea Nymphaea Nuphar Barclaya Barclayaceaea Brasenia Cabombaceaea Cabomba Amborella AmboreliaceaeC 0 Schisandra Schisandraceaef F IlUicium Ifllciaceaef Austrobaileya Austrobaileyaceae r1 Chloranthus Chloranthaceaed Aristolochia Saruma Aristolochiaceaee

Houttuynia Saururaceaed Saururu~s Peperomia Piperaceae d monocots Hernandia HernandiaceaeC Hedyearya Monimiaceaec Idiospermum IdiospermaceaeC Calycanthus chinensis7 Calycanthusfloridust CalycanthaceaeC Chimonanthus Cinnamomum ZLauraceaeC Persea Canella Canelaceae b Drimys zlitrcab

Magnolia macrophyllat Magnolia salicifolia Michelia Manglietia Talauma singaporensis Magnoliaceae b Talauma ovatat Magnolia tripetalat Magnolia hypoleuca 0 Liriodendron W Degeneria Degeneriaceae b Asinona Annonaceae

Galbulimima Himantandraceaeb Eupomatia Eupomatiaceae b Knema Myristicaceaeb Ceratophyllum Ceratophyllaceaea sst I ast II ast m ast IV ast V ros mE aMagnoliidae, Nyphaeales rca I1 rca I Magnoliidae, Magnoliales ham Laurales ham III CMagnoliidae, ran Magnoliidae, Piperales amon Magnoliidae, Aristolochiales lan mag JMagnoliidae, Illiciales cer gne r- - pin con cyc FIGURE 4. Basal portion of the overall analysis showing the positions of Ceratophyllaceae (inaperturate pollen), monocots (uniaperturate pollen), eudicots (dicots with triaperturate pollen), and the three clades of "primitive" dicots (monosulcate pollen). Note that, exclusive of Ceratophyllum, the angiosperms form two sister groups marked by the general pollen aperture number (one versus three). (Numbers above the branches in B are the numbers of substitutions optimized onto one tree randomly selected from the 3,900 saved in Search II.) Genera marked with a "t" in A were omitted from Search II; genera marked with an asterisk in B were not available for Search I. The "Canella" in this figure represents the position of two sequences amplified from a total cellular DNA template (see Materials & Methods). Note that A is the consensus tree of Search I, whereas B is a single tree with branches not present in the strict consensus of Search II marked by an arrow. Volume 80, Number 3 Chase et al. 557 1993 Phylogenetics of Seed Plants

4B 18 eudicots 6Victoria 4 ~& Nymphaea Nymphaeaceae a 4 Nuphar a 0 Barclaya Barclayaceae a 12 10 6Brasenia : Cabombaceae a Cabomba0 35 6 14 Amborella Amborellaceaec 5 15 Schisandra Schisandraceae P 4 14 IlliciuzmM Iliiciaceae f 24 Austrobaileya Austrobaileyaceae - 14 12 Hedyosmum*d Chloranthus Chloranthaceaed Sarcandra* 43 Canella Canellaceaeb 7~~~~~ Drimysb 5 = ~ Beliolum Winteraceaeb Tasmannia* 2Magnolia salicifolia

22 l l | r 6 Talaumaovata MMagnolia hypoleuca 1 b 6 -1 Manglietia Magnoliaceae 6 6 8 Talaumasingaporensis 2 34 8BLiriodendrontulipifera _f2 Liriodendronchinensis* 13 ~~~~~~~~~~bW 1 > 4 3 r Degeneria Degeneriaceae 24 Galbulimima Himantandraceaeb 7 | _ EApomnatia Eupomatiaceaeb 7 7 ~~~022Annona 1 22 Asimina Annonaceaeb 01 21 Cananga* Knema Myristicaceaeb 13 Cinnamomnum Luaee 59 3 Persea c Hedycarya Monimiaceaec 15 Idiospermum IdiospermaceaeC a 8 9 Calycanthus 7 Calycanthaceae Chimonanthus 10 33 Hernandia jj] Gyrocarpusmonocots Hernandiaceae Saruma Aristolochiaceae e

12 ~3 SAristolochiaLactoris* Lactoridaceaee O

Houttuynia iSu aee Piper* Pp aad Peperomia Piperaceae Ceratophyllum Ceratophyliaceaea ast L -ast II W astlm IV { o Magnoliidae, Nyphaeales ?0 ros111Magnoliidae, Magnoliales s .n Magnoliidae, Laurales ran Magnoliidae, Piperales , m --agrt Magnoliidae, Aristolochiales paFlI Magnoliidae, Illiciales

d ! P~~~~~giin cyc 558 Annals of the Missouri Botanical Garden

5A commelinoids Xanthorrhoea XanthorrhoeaceaeJ Kniphofia Aloe Haworthia J AsphodelaceaeJoeaee Sansevieria DracaenaceaeJ Danae RuscaeaeJ Nolina 7 Nolina findheimnerinajreecurvata NolinacaceaeJ Bowiea HyacinthaceaeJ Chlorophytum AnthericaceaeJ Clivia AmaryllidaceaeJ riaee _ E ~~~~Anomatheca Cyanastrum TecophilaeaceaeJ CureuigH HypoxidaceaeJ Neuwiedia Orchidaceae' OncidiumSmilax Smilacaceaee r-LiliumMedeola Liliaceae'Llaee Alstroemeria Alstroemeriaceae' CokchicumI Burchardia Colchicaceae' Chamaelirium Melanthiaceaeb Vellozia Velloziaceaeh Freycinetia Pandanaceaeg Cyclanthaceaef = Iwgoseorea Dioscoreaceaee Tacca Taccaceaee Aletris Melanthiaceaeb Burmanniat Burmanniaceaed _ _ ~~~~~~~~Sagitarwagraminea- Sagittarialatifa Alismataceavc _ ' Alsm ar Potamogeton Potamogetonaceaec r Pleea Melanthiaceaeb " eSathiphyllum ast I Araceaea ast ymnostachys ast IIIf ast IV . st V Aranae _ rosm bLilianae, Melanthiales rosI cAlismatanae { hamnII tLilianae, Burmanniales ran 'Lifianae, Dioscoreales _ _Cyclanthanae _ lau~~O gPandananae Iaga . crg lkBromelianae,Velloziales JLgne 'Lilianae, Liliales con JLilianae, Asparagales

FIGURE 5. The five basalmost lineages of the monocots, composed of the aroids, alismatids, and lilioid groups. (Numbers above the branches in B are the numbers of substitutions optimized onto one tree randomly selected from the 3,900 saved in Search II.) Genera marked with a "t" in A were omitted from Search II; genera marked with an asterisk in B were not available for Search I. Note that A is the consensus tree of Search I, whereas B is a single tree with branches not present in the strict consensus of Search II marked by an arrow. Volume 80, Number 3 Chase et al. 559 1993 Phylogenetics of Seed Plants

5B 198 Cypripedium* Neuwiedia Orchidaceaed 27 Oncidium 19 Lomandra* Dasypogonaceae 5 s 17 38 Hemerocaifis* Hemerocallidaceae' 1L7 Chlorophytum Anthericaceae' 6 21 Xanthorrhoea Xanthorrhoeaceae 25 6 Kniphofia 13 Aloe Asphodelaceae' 10 1 Haworthia

18 Scilla* Hyacinthaceae 12 10 21 BowieaSansevieria 2 t Dan~~~~~B~ae Ruscaceae' 2~~~~~~~~ 11 | Nolina Nolinaceae Le 16 21 Clivia Amaryllidaceae' 3 1612 21iris d R/19 19 Anomatheca jIridaceae 23 Cyanastrum Tecophilaeaceae 2 18 Curculigo Hypoxidaceae 6 Hypoxis 4 commelinoids A 27 Dioscorea Dioscoreaceaed 3 5 Tacca Taccaceae d 1 9 Pandanus* Pandanaceaeh I14lALFreycinetia10 Sphaeradenia Cyclanthaceaeeg Vellozia 30 Velloziaceae Smilax Smilacaceae d Calochortus* Calochortaceaed is 15 10 *13 ~~~~~~~~~1714Lilium d 1;0Medeola 1Liliaceae ii 24 18Cokhicum 12 23 16 Colchicaceaed Burchard1aBuchrdad 36 Astroemeria Alstroemeriaceae 9 ry-i-3 Veratrum* 8 57 Chamaelirium MelanthiaceaeC 22 Aletris 18 Pleea 3 8 Sagittariagraminea 4 20 Saglttarialatifolia Alismataceaeb 24 Alisina b_ 13 Potamogeton Potamogetonaceae 41 Spathiphyllum Araceaea Pistiai5 I a Lemna* Lemnaceae 45 Gymnostachys ] Araceaea Acorus* ast I ast U ast III ast IV a astV Aranae ros I rosm Alismatanae C can Lilianae, Melanthiales ham I Lilianae, Liliales rane _ palII eLilianae,Dioscoreales nmagf X Mon < fBromelianae, Velloziales i [ ~~palI gCycIanthanae rl gne ~~~~~hPandananae 11 ' ~~~~- pin i ~Lilianae,Asparagales cyc 560 Annals of the MissouriBotanical Garden

6A Avena Puccinellat Triticum Hordeumt Aegilops Poaceae' Cenchrus _ ~~~Pennisetumt Neurachne Oryza Efegia Restionaceaei Juncus 7 uccee Oxychloe Juncaceaei YPerus CyperaceaeJ Prionium Juncaceaej Flagellaria Flagellariaceae' ,Typa Typ aeh Stegolepis Rapateaceaef Aechmeat Ananas Glomeroptitcairniat CatopsisT Bromeliaceaeg Tillandsia Puya Hechtiat Tradescantiapallida Tradescantiazebrina Commelinaceaef Tradescantiasoconuscana Pontederia Pontederiaceaee Philydrum Philydraceaed Anigozanthos Haemodoraceaec Ravenala Strelitzia Strelitziaceae Phenakospermum MarantaC Marantaceaeb HedychiumRiedelia Zingiber Zingiberaceaeb Globba Costus Csaee Tapeinocheilos Costaceaeb _ierMusaceaeb Orchidantha Lowiaceaeb Heliconia Heliconiaceaeb Caryotat Phoenixt

minmm inmmm ast I fhmm~ DrymophiloeusChamaedoreaI Arecaceaea ast II Calamust ast III Serenoa ast IV ast V aArecanae ros IV b ros III Zingiberanae ros I CBromelianae, Haemodorales -ham I Philydrales hamlI dBromelianae,e ran Bromelianae, Pontederiales Pal fCommefinanae, Commelinales one n ~~~cmerg hBromelianae, Typhales g.[1 pin 'Commelinanae, Poales con JCommelinanae, Cyperales cyc FIGURE6. The terminallineages of the monocots,composed of the palms,gingers, and commelinoids.(Numbers above the branches in B are the numbers of substitutions optimized onto one tree randomly selected from the 3,900 saved in Search II.) Genera marked with a "t" in A were omitted from Search II; genera marked with an asterisk in B were not available for Search I. Note that A is the consensus tree of Search I, whereas B is a single tree with branches not present in the strict consensus of Search II marked by an arrow. Volume 80, Number 3 Chase et al. 561 1993 Phylogenetics of Seed Plants

6B 13 Avena 11 Triticum 5 61 Aegilops 61 21 11 Cenchrus Poaceae 23 Neurachne 13 13 Oryza 9 Bambusa* 1 464 Lachnocaulon* Eriocaulaceaef _ 33 Elegia Restionaceaei FlageUaria Flagellariaceae1

8 2 Juncaceae 7 10 Oxychloe | g 2 Cyperus Cyperaceae w IL ~~Carex i lo 3 r 31 Prionium Juncaceae 11 Sparganium* Sparganiaceae Typha Typhaceaeh Stegolepis Rapateaceaef 17 Tillandsia 432 - A~unanas Bromeliaceae g

32L-Puya 2 9 Commelina* 8 7 Tradescantiapallida Comfelinaceae 25 2t31 Tradescantiazebrina 29 Tradescantiasoconuscana 16 29 Pontederia Pontederiaceaee 15 Philydrum Philydraceaed 3 Anigozanthos HaemodoraceaeC 10 Ravenala 1 6 Strelitzia Strelitziaceaeb Phenakospermum

| | ~~~93 aa~ Marantaceab V Calathea Marantaceae 6 11 9Hedychium 20 Riedelea Znieaaeb 12 ZingiberZigbrca 5 ~~Globba

282 Tapeinocheilos 11 11 Musa Musaceaeb - Orchidantha Lowiaceaeb 22 Heliconia Heliconiaceaeb 6 Chamaedorea 13 Drymophloeus Arecaceaea Nypa I astl Serenoa -ast lIla ast IVI astIV aArecanae ros I b rsI Zingiberanae r caam cBromelianae, Haemodorales Un ~~d ,la Hm dBromelianae, Philydrales rpaanl I1 Bromelianae, Pontederiales m ag Commelinanae, Commelinales ff . .~ palI gromelianae, Bromeliales IL cer h~~~Bromelianae,Typhales ril pin i ~~Commelinanae,Poales l cgynC j~~~Commelinanae,Cyperales 562 Annals of the Missouri Botanical Garden

7A r higher eudicots TrochodendronTrochodendraceae Tetracentron Tetracentraceae , Sabia Sabiaceae' Lambertia Proteaceae e Nelumbo Nelumbonaceae | Platanus Platanaceaeb Akebia Lardizabalaceaec MahoniaBreidcam CaulophyllumBerberidaceae Ranunculus Xanthorhiza Ranunculaceae c Caltha Cocculus Menispermaceaec OW Euptelea Eupteleaceae b Dicentra Fumariaceae' PapajjPPapaveraceaejP Sanguinari _r t I _t 9III Mt IV Fa- IV Magnoliidae, Papaverales rag~ Ma ragmil 1Hamamelidae, Hamamelidales Im Magnoliidae, Ranunculales i X au d Magnoliidae, Nymphaeales A - u"re Rosidae, Proteales r nCYC Hamamelidae, Trochodendrales FIGURE7. The basalmostlineages of the eudicots,composed of Ranunculales-Papaverales,Trochodendrales, and a heterogeneouslineage (plus Gunnerain B). (Numbersabove the branchesin B are the numbersof substitutions optimizedonto one tree randomlyselected from the 3,900 saved in Search II.) Generamarked with an asteriskin B were not availablefor Search I. Note that A is the consensustree of Search I, whereasB is a single tree with branchesnot presentin the strict consensusof SearchII markedby an arrow. Volume 80, Number3 Chase et al. 563 1993 Phylogenetics of Seed Plants

7B h hgher eudicots Gunnera Gunneraceae'h 8 20 15 TrochodendronTrochodendraceaeg 5

1 Tetracentron Tetracentraceaeg 4 1 31 Pachysandra* Buxaceaef 0 10 42 Sabia Sabiaceae'C Lambertia Proteaceaee 6Nelumbo Nelumbonaceaed 13Platanus Platanaceae~~~~~~~b -N 32 Akebia Lardizabalaceaec 18 27 Mahonia 8 15 26 Caulophyllum Berberidaceae

1 1025 Ranunculus 24 6 80 Xanthorhiza Ranunculaceaec 21 Caitha M 29 Cocculus Menispermaceae _ 19 Euptelea Eupteleaceaeb 7 12 Dicentra FumariaceaL4 39 11 Papaver 1 . Sanguinaria Papaveraceae' AI ~~~a A[S^^M bMagnoliidae, Papaverales IrotVbHamamelidae, Hamamelidales j r Su1 C r l r~sm Magnoliidae,Ranunculales -ham:am I0I Magnoliidae, Nymphaeales pagie Rosidae Proteales Mon f - -141 fRosidae, Euphorbiales ._ Trochodendrales pumptyRieHlalh Hamamelidae, -----CYCRosidae, Haloragales 564 Annals of the MissouriBotanical Garden

8A Boykinks Saxifraga Saxifragaceae' Astilbej Heuchera Tetracarpaea Grossulariaceaec Myriophyllum Haloragaceae d Penthorum Saxifragaceaec Sedum Dudleya Crassulaceaec Kalanchoe Crassula Ribes Grossulariaceaec

nRhodoleja Hamamelis CHamamelidaceae [Cercidiphyllum Cercidiphyllaceaeb asttl Daphniphyllum Daphniphyllaceaea ostIV ust V ros IV ro I

mo{Cbn Hamamelidae, Hamamelidales rmery dRosidae, Rosales ipin ine Rosidae, Haloragales cyc

FIGURE8. The basalmost lineage of rosid dicots, which includes a number of lower hamamelids. (Numbers above the branches in B are the numbers of substitutions optimized onto one tree randomly selected from the 3,900 saved in Search II.) Genera marked with a "t" in A were omitted from Search II; genera marked with an asterisk in B were not available for Search I. Note that the positions of this clade and that of the Caryophyllidae (Fig. 9A, B) differ significantly in the results of Searches I and II (see Figs. 1, 2). Note that A is the consensus tree of Search I, whereas B is a single tree with branches not present in the strict consensus of Search II marked by an arrow. Volume 80, Number3 Chase et al. 565 1993 Phylogenetics of Seed Plants

8B 8 Pterostemon Grossulariaceae 10 Itea , 1 Boykinia _1 Heuchera Saxifragaceae' 5 Saxzfraga 21 Grossulariaceae' 12 3 Tetracarpaea 4 s Myriophyllum Haloragaceae 15 Penthorum Saxifragaceae 1 54 Paeonia Paeoniaceaed 16 18 Sedum I 10 25 9-,12 Dudleya JCrassulaceaeC 25 Kalanchoe Crassula 5 22 Ribes Grossulariaceae' 8 Rhodoleia Hamamelidaceae a 12 a 2 Cercidiphyllum Cercidiphyllaceae ' 3 Daphniphyllu Daphniphyllaceae 2 Hamamelis :sSIt _I11 Altingia* Hamamelidaceaea astIV Liquidambar*- _ ros I _ rosIII gun rosid III hamI ran aHamamelidae,Hamamelidales rl agI 'Hamamelidae, Daphniphyllales ' lau'mn CRosidae, Rosales cerd cn1 Dilleniidae, Dilleniales con e gin Rosidae, Haloragales 566 AnnalsMissouri ofBotanical the Garden

9A Dillenia Dilleniaceae' Dianthus Caryophyllaceae' SApinpaleix Chenopodiaceaee Amaranthus Amaranthaceaee Basella Basellaceae' Portulaca Portulacaceae Alluaudia Didiereaceae' Mollugo Molluginaceae Phytolacca Phytolaccaceaee Mnirabilis Nyctaginaceae Rivina Phytolaccaceaee Trianthema Aizoaceae Stegnosperma Phytolaccaceaee Nepenthes Nepenthaceaeb Plumbago Plumbaginaceaed Rheum Polygonaceaec Drosera Droseraceaeb

atI Vitis Vitaceaea ast I

IVII MtMtIV rosid III ros IV ros ros III a ros Rhamnales hamIIII b Rosidae, r-ham I Dilleniidae, Nepenthales pal C0 inion Caryophyllidae,Polygonales G Me~g dCaryophyllidae,Plumbaginales

pin Caryophyllidae, Caryophyllales IYC ccynDilleniidae, Dilleniales

FIGURE 9. The lineage that includes the Caryophyllidae. (Numbers above the branches in B are the numbers of substitutions optimized onto one tree randomly selected from the 3,900 saved in Search II.) Genera marked with an asterisk in B were not available for Search I. Note that the positions of this clade and that of rosid IV (Fig. 8A, B) differ significantly in the results of Searches I and II (see Figs. 1, 2). Note that A is the consensus tree of Search I, whereas B is a single tree with branches not present in the strict consensus of Search II marked by an arrow. Volume 80, Number3 Chase et al. 567 1993 Phylogenetics of Seed Plants

9B 30 21 25 Dianthus Caryophyllaceae' 21 Atriplex ]Chenopodiaceae 19 Amraranthus Amaranthaceae e 5 e 5 77Basella 30 Basellaceae 6 Portulaca Portulacaceae e 10 Alluaudia Didiereaceaee 16 Mollugo Molluginaceaee 11*19 Phytolacca accaceae Phyto e 0 14 229 Mrabilis Nvcta inaceae 25 Rivina Phytolaccaceaee 26 anthema Aizoaceaee s Stegnosperma Phytolaccaceaee 14u31 Plumbago Plumbaginaceaec 24 Rheum Polygonaceae 9 Nepenthes 1 15 1 epenthaceae' 20 Droserafiliformis 3 Drosera spathulata* b Drosera etiolaris* roseraceae 39Dionaea 58 Phoradendron Viscaceaea 110Schoepfia Olacaceae' _gassI Osyns Santalaceae' ast III ant TV MntV roi car, caryophyllids n Ib Rosidae, Santalales pslIIDilleniidae,mn Nepenthales ,,,sgnw Caryophyllidae, Polygonales IMI palI Caryophyllidae, Plumbaginales _~~~~m e G- fin >Caryophyllidae, Caryophyllales cyc 568 Annals of the Missouri Botanical Garden

10A Gossypium Malvaceaej Leitneria Leitneriaceae' Ailanthus Simaroubaceaeh Poncirus Rutaceaeh Bursera Burseraceaeh Acer Aceraceaeh Cupaniopsis Sapindaceaeh Schinus Anacardiaceaeh Tropaeolum Tropaeolaceaea Brassica Brassicaceaee Capparis Capparaceaee Reseda Resedaceaee Tovaria TovariaceaeC Batis Bataceaeg Linnanth Limnanthaceae Floerkea Carica Caricaceaef Moringa Moringaceaee Qualea Vochysiaceae Clarkia Oenothera Epilobium Hauya Onagraceaec Fuchsia t Circaea Ludwigia Lythrum Lythraceae Quisqualis Combretaceaec b Crossosoma Crossosomataceae Pelargonium Geranium Geraniaceae Monsonia M~stII Greyia Greyiaceaeb at~stm11Francoa Saxifragaceaeb J ast IV Viviania Geraniaceaea _ros IV rosIII rosidlI

hr I aRosidae,Geraniales fDilleniidae, Violales , bRosidae,Rosales 9Dillenfidae,Batales l cer cRosidae,Myrtales hRosidae, Sapindales gne dRosidae,Polygalales 1Hamamelidae, Leitneriales _ ~~~~~pin con eDilleniidae,Capparales JDilleniidae, Malvales

FIGURE10. One of the two "higher" rosid lineages. (Numbers above the branches in B are the numbers of substitutions optimized onto one tree randomly selected from the 3,900 saved in Search II.) Genera marked with a "t" in A were omitted from Search II; genera marked with an asterisk in B were not available for Search I. Note that A is the consensus tree of Search I, whereas B is a single tree with branches not present in the strict consensus of Search II marked by an arrow. Volume 80, Number 3 Chase et al. 569 1993 Phylogenetics of Seed Plants

10B 13 Leitneria Leitneriaceae 15 1 Ailanthus Simaroubaceae c 10 4 Poncirus Rutaceae c 24 21 21 Bursera Burseraceae c 17 Acer Aceraceae c 19Cupaniopsis Sapindaceae c Schinus Anacardiaceae c Bombax* Bombacaceae h _2 r 2214 Tilia* Tiliaceae h 1 10 Theobroma* Sterculiaceae h 4 10T 12 Thespesia* Malvaceae h 40 _ Shorea zeylanica* pterocarpaceae g 12 Shorea stipularis* Di 7 1- Moringa Moringaceae d 14 Carica Caricaceae f 2 Stalya Brassicaceae d 6 16 Brassica

7 6 12 Coapris* Capparaceae d ~9 38 -Cpai 1 16 Reseda Resedaceae d 16~~2 Tovaria Tovariaceae d 12 _Koeberlinia* Capparaceaed 814 1 Batis Bataceae e _ 20 31 Limnanthes L 44 Floerkea Setchellanthu$* Capparaceae d 5L Bretschneidera* Bretschneideraceae c 10 -- Akania* Akaniaceae c Tropaeolum Tropaeolaceae a 2525 Crossosoma Crossosomataceae b 0 Pelargonium I26I~i2Fi~Geranium Geraniaceae a astI ast 11 33 Monsoniaa~soi -tast InI Hypseocharis* Oxalidaceae a ros I rosid II bRosidae, Geraniales bRosidae, Rosales ham I c Rosidae, Sapindales pmal eDilleniidae, Capparales .mon Dilleniidae, Batales lauf pal I Dilleniidae, Violales ner . - conh8~~ne 9Dillenfidae, Theales pin Dilleniidae, Malvales gin Hamamelidae, Leitneriales IcycHaaeiaLinrls 570 Annals of the Missouri Botanical Garden

11A Celtis Ulmaceae Morus Moraceael Rhamnus Rhamnaceaen Krameria Krameriaceae' Guaiacum Zygophyllaceaem Spiraea Photinia Roaeeb Prunus Rosaceae Geum Myrica Myricaceae? Fagus Fagaceae J Casuarina Casuarinaceaek Chrysolepis Fagaceaei Datisca Octomeles DatiscaceaeDaiscca Trigonia C Licania Chyoaaaeeb Chrysobalanus~IrYsobalanaceae Erythroxylum Erythroxylaceaeh Viola Violaceae f rg~ Ochna Ochnaceaei Drypetes Euphorbiaceaeg Humiria Humiriaceaeh Euphorbia Euphorbiaceaeg Passiflora Passifloraceaef Acridfocarpus Galphimia7 Thryallis Bunchosia MalpighiaceaeC Mascagnia Dicella Byrsonima . uonymus Celastraceaee * Brexia Grossulariaceae Lepuropetalon Saxifragaceaeb Kirengeshomat Hydrangeaceaeb Parnassia Saxifragaceaeb Pisum Medicago Fabaceaed Albizia Bauhinia Securdlaca Polygalaceaec Cephalotus Cephalotaceaeb Platytheca Tremandraceaec Bauera 1Cunoiceeb Ceratopetalumj noniaceae Eucryphia Eucryphiaceaeb Oxalis Oxalidaceaea ast I ast H ast IlI ast IV ast V rosid I _ r rosos SI aRosidae, Geraniales ros b Rosidae, Rosales Dilleniidae, Theales .ham II CJ ham I Rosidae, Polygalales d Hamamelidae,Fagales palin Rosidae, Fabales k Hamamelidae, Urticales r { flaun efRosidae, Celastrales Hamamelidae, Casuarinales mearg Dilleniidae, Violales mRosidae,Sapindales rll in gRosidae,Euphorbialesn Rosidae, Rhamnales c~cy~n Rosidae Linales Hamamelidae,Myricales FIGURE 11. The second "higher" rosid lineage. (Numbers above the branches in B and C, see foldout, are the numbers of substitutions optimized onto one tree randomly selected from the 3,900 saved in Search II.) Genera marked with a "t" in A were omitted from Search II; genera marked with an asterisk in B and C were not available for Search I. The taxon labeled "Kirengoshoma" was, subsequent to Search I, discovered to be misidentified and was removed from Search II; its identity is unknown. Note that A is the consensus tree of Search I, whereas B and C are a single tree with branches not present in the strict consensus of Search II marked by an arrow. llC 2 2 other rosid I b Cephalotus 10 6 25 Platytheca TremandraceaedCephalotaceae Bauera cunoniaceaeb Eucryphia Eucryphiaceaeb

15 O1 xverrhoa* Oxalidaceae a Oenothera 16 Clarkia 2 9 1 31 Epilobium 4 24 Circaea Onagraceae 8 17 1 ~~Lopezia 188 Hauya 12 Ludwigia 10 3 Lythrum Lythraceaec 3 Trapa* Trapaceaec 1 16Punica* Punicaceae c 2 _5 21315 Heteropyxis* Myrtaceaec 12 31 Qualea Vochysiaceaed 6 Mouriri ] Melastomataceaec 19 Quisqualis ICombretaceaec 9 11Terminalis*'_ 4 16 Greyia Greyiaceaeb 1 ~ - Francoa Saxifragaceaeb 29Viviani WeVivandia 1Geraniaceaea rosid II astI ast III ast IV ros rosid I (in part)

h Geraniales _amaIran Rosidae, rcpikgIIal bRosidae, Rosales

paE'uCl Rosidae, Myrtales cer dRosidae,Polygalales pin gin cyc Volume 80, Number 3 Chase et al. 571 1993 Phylogenetics of Seed Plants

11B 5alph imia liB 6 10 Acridocarpus 2 1 Mascagnia Malpighiaceae B vronima _ Euphorbiaceae' 407 ;Pshorbja 4 40 Passiflora Passifloracraee 12 Ochna Ochnaceae 37 Ham''ia Humiriaceae0 8 _14 10 Trigonia _ Trigoniaceae k Chrysobalanus jChrysobalanaceae g e22 aReinwardfia* Linaceae? 11 ^Viola Violaceaee 69?Erythroxylam Erythroxylaceae? 18 tes Euphorbiaceaen Dryemus Celastraceaem 31 21 21 Brexpa Grossulariaceae g Lepuropetalon zSaxifragaceaeg 28 Parnassa Jfairaaee 1 9 35 Sargentodoxau Sargentodoxaceaef 2 ~~~~15L947 Pisum 14MedIi 16 ago Fabaceael 12 28 Albizzia. 32 r 23 Bauhinia =-3 Securidaca Polygalaceae' 3Huulus* Cannabaceaed

] Ulmaceae d 5 16 20 Trema* 12 19Boehmeria' Urficaceaed MOM'] Moraceaed 6g Rhamnus Rhamnaceae 3 2 21 469 Krameria Krameriaceaei 9220 2 Guaiacum Zygophyllaceaeh 9 Phofinia Pp15Siraea Rosaceaeg 16 GeumfCoriaria* Coriariaceaef 13 15 1 Octomeles 4 ~~~~~~Tehrameles* Daisaee 4 , 11 Datisca cannabina* Datscaceaee 9 3 Datisca glomerata _ 28 uffa* 12 8 14 Cucurbita* Curcurbitaceaee 7 32~~~~Cucumis* Begoniaceae. 188 Legonia* 8 7 BMric MyricaceaeK ,1 Betula* BetulaeB 7 7 21 Casuarina Casuarinaceaec 11 14 Carya* Juglandaceaeb 3 _ ffi~~1Fagus t1 20 Trigon lanus* Fagaceaea astfl 20 8 Nothofagus balansae* astIV Nothofagus dombeyi* roI< ro ros~rM rosid I (in part) am U Hamamelidae, Fagales Rosidae, Polygalales ran Hamamelidae, Juglandales k Rosidae, Rhamnales II C rp{al Hamamelidae, Casuarinales Hamamelidae, Myricales mon Hamamelidae, Urticales Rosidae, Fabales pal I Dilleniidae, Violales mRosidae, Celastrales -1 1 c~~gene fMagnoliidae, Ranunculales "Rosidae, Euphorbiales rl t ~~~pin h Rosidae, Rosales ?Rosidae Linales I gin R~~~~osidae,Sapindales PDilleniidae, Theales 572 Annals of the Missouri Botanical Garden

12A higher asterids Cornuskousa Cornus canadensis Cornuswalteri Cornaceaed Cornus mast Cornusflorida A a Alangium Alangiaceaed V: _ ~~Decumaria Carpenteria *S Deutzia Hydrangeaceaef|C Phi ladeiphus -0 Hydrangea - Nyssa 7Nssaceae d Camptotheca Diplopanax Araliaceae' Davidta Nyssaceae d Gunnera Gunneraceae c Paeonia Paeoniaceae b Phoradendron Viscaceae"a Schoepfia Olacaceaea Osyris Santalaceaea _ast m

r[-ros ros 1IV b Rosidae, Santalales rho~sIII Dilleniidae, Dilleniales

pan Rosidae, Haloragales Cornales roMe mage Rosidae, ero. f Rosidae, Apiales pin ~ ~ ~ os rLC e~n Rosidae, Rosales

FIGURE 12. The two basalmost lineages of the general asterid clade. (Numbers above the branches in B are the numbers of substitutions optimized onto one tree randomly selected from the 3,900 saved in Search II.) Note the different composition of asterid V in Searches I and II. Species marked with a "t" in A were omitted from Search II. Note that A is the consensus tree of Search I, whereas B is a single tree with branches not present in the strict consensus of Search II marked by an arrow. Volume80, Number3 Chase et al. 573 1993 Phylogenetics of Seed Plants

higher asterids

26 Cornus kousa 12 7 1 26 Cornus 61 canadenssCornaceae'Crnca 11 4 Cornuswaiteri Cornusflorida 27 Alangium Alangiaceae'

5 X Nyssa ] Nyssaceae'd 1 Davidia -.w 8 ~ Diplopanax Araliaceaee 10 Camptotheca Nyssaceae d

42 Decumaria 12 12 Hdrangea 2k3 yCarpenteria jHydrangeaceae' 1 Philadelphus I 21 Deutzia I 6 4Dillenia Dilleniaceaeb | Vitis Vitaceae a ast I ast II ast III~ astV r ast C" Rosidae, Rhamnales unI b

-ran Dilleniidae, Dilleniales Moagnmond dRosidae,Rosales j1 ~cearle Rosidae, Cornales pin Rosidae, Apiales 574 Annals of the MissouriBotanical Garden

13A Clavija Theophrastaceae Anagallis PrimulaceaeJ Ardis'a Myrsinaceae3 Diospyros Ebenaceae b { Diapensia Diapensiaceae' Poliemonium Polemoniaceae h E Symplocus Symplocaceae b __ Impatiens Balsaminaceaeg Camellia Theaceae f Calluna t Erica Befaria Ericaceaec Elliottiat Leiophyllumt CeratiolaEmpetraceaec Rhododendron Cassiope Chamaedaphne Zenobiat Ericaceaec Daboecia Leucothoet Gaultheria Vaccinium Cyathodest Pentachondra Leucopogont Epacridaceaec Dracophyllum t Epacris Arbutus Eiaee ,______{____ Arntostaphylos Ericaceae Pyrola Pyrolaceaec Enkianthus Ericaceaec f _~ I Actinidia Actinidiaceae Sarracenia Sarraceniaceae' Roridula Byblidaceaed Cyrilla Cyrillaceaec SStrax Styracaceae b E Cethra Clethraceaec Ma~nilkara 1Sapotaceaeb Chryso hyllumz aoaee Fouquiena Fouquieriaceae'

H d asltasttm asterid III ast IV a Violales astYV b Dilleniidae, s IV roslfl c Dilleniidae, Ebenales ros d Dilleniidae, Ericales H -- hamdm Rosidae, Rosales ran Dilleniidae, Nepenthales - w:moln f Dilleniidae, Theales a.g gRosidae, Geraniales cer hAsteridae, Solanales - pinc~o iDilleniidae, Diapensiales cYc Dilleniidae, Primulales

FIGURE 13. The immediate sister lineage to the clade composed of traditional asterids. (Numbers above the branches are the numbers of substitutions optimized on the general semi-strict consensus tree in A and one tree randomly selected from the 3,900 saved in B.) Genera marked with a "t" in A were omitted from Search II; genera marked with an asterisk in B were not available for Search I. Note that A is the consensus tree of Search I, whereas B is a single tree with branches not present in the strict consensus of Search II marked by an arrow. Volume 80, Number 3 Chase et al. 575 1993 Phylogenetics of Seed Plants

13B 31 17 26 Clavijal Theophrastaceae j 5 1 Anagallis Primulaceae i . 24 Ardisia Myrsinaceaej L.L6fl Styrax Styracaceae e 32 Clethra Clethraceae f 3 ri__ Symplocus Symplocaceaee 18 Diospyros Ebenaceaee 6 15 CassiopeErceaf 13 2.3 BefariaEraee 42 Ceratiola Empetraceaef 3 Erica 8 18 Rhododendron ' 1 Chamaedaphne Ericaceaef 18Gaultheria 15 12 11Daboecia Vaccinium 201rPentachondra - _ 3 .4 Epacris Epacridaceaef 23 Pyrola Pyrolaceaef 24 Aribtcs Ericaceaef 11 Enkkanthus 2 Heliamphora* Sarraceniaceae 3 3 16 19 ~ 2Roridulaanilkara Byblidaceaeh 10 3 ~~~~~~~Camellia Theaceae g 16Cyrlla19 Manilkara FouqeCyrillaceaea --PTleActnidia ]SapotaceaeeActimniiaceaeg 41 DlhrYSOPhYllum d 9L I bImpatiens Balsaminaceae 4rll2 Diapensia Diapensiaceae 14m dPolemonium, Polemoniaceae b Fouquieria FouquieriaceaeEa ast I ast HI h~osiasterid III ast HIIa ast IV ast V aDilleniidae, Violales ros rosIIISlnaeI1 bAsteridae,Soa le -ar ~~cDilleniidae,Diapensiales Geraniales -ham IdRosidae,e ran Dilleniidae, Ebenales magf Mon lau DilniaEricales

cer gDilleniidae, Nepethle s ~~cyc 'Dilleniidae, Nprimulales 576 Annals of the Missouri Botanical Garden

14A SYmphoricarpos Caprifoliaceae Valeriana Valerianaceae Dipsacus Dipsacaceaei Sambucus Caprifoliaceaei Adoxa Adoxaceael ViburnumCaifoliaceae Boopis Ca yeraceaeh Cameliana Campanulaceaeg Scaevola Goodeniaceae g Stokesiat Pipto carp ha _ ' Vernonia Gerbera Traopogon _ _r Cic orium Lactucat Gazaniat Cacosmia Echinopst Carthamnus Dimorphotheca Felicia Achillea Asteraceae f Chrysanthemumt BlennospermumtSenecio _ FC P~~laveriat Chromolaena Coreopsist Eupatorium He janthust Barnadesiat ______asy hylum Menyanthsa Menyanthaceaee Corokia Cornaceaec Hedera A raliaceaed Pittosporum Pittosporaceae b Coriandrum Apijm Cboniumt |Apiaceaed Sanicula Griselinia Cornaceae c Berzelia Bruniaceae b Escallonia Grossulariaceaeb Helwingia CornaceaeC Phyllonoma Grossulariaceae b Hex vomitoriat lquifoliaceaea ast I Ilex crenata ast H 00 -astast MII uSastvIVasterid II roslIV rosm aRosidae, Celastrales rosfl b rosI Rosidae, Rosales hamI CRosidae, Cornales pran Rosidae, Apiales ront~laeAsteridae, Solanales mag f Asteridae, Asterales gne g~tei eCampanulales

con ~~~~hAsteridae,Calycerales CYC~~~~ 'Asteridae, Dipsacales Volume80, Number3 Chase et al. 577 1993 Phylogenetics of Seed Plants

1 14B Adoxa Adoxaceaei 12 Sambucus 9 14 Viburnum Caprifoliaceaei 14 Symphoricarpos Valeriana Valerianaceae' 14 Dipsacus Dipsacaceaei 4 9 9 Hedera Araliaceaeh

Apiaceaeh 4______4 16 Apium 13- PittospaorumCoriandrum Pittosporaceaeb 4 211 21 Griselinia Cornaceaec ~~11 17 261410Aralia SaniculaBerzelia Bruniaceaeb 8 rITI_.Boopis Calyceraceae d 28 Scaevola Goodeniaceaed 19Dimorp hotheca 26 Felicia < ,622Achillea Asteraceae f 9 1~~~3Senecio 2 6 EscaChromolaena 23 416 Eupatorium3Tagetes 8 10 Gerbera 8 4 6 5Vernonia 11 11 Piptocarpha 13 Cacosmia 6 8v I 3' 8 ______Cart 33 hamnus 1Corokia Cornaceaec 14 14 Villarsia Mnatceae 40 Menyanthes Meynhce 3 IV31Lobelia rhanm23 d steiCampanula le 20 Escallonia Grossulariaceaea 13L gnr5 Heiwingia Cornaceaec b l pinRosieGrossulariaceae 22Phylloam Ilex ~~Aquifoliaceaea ast I MtastH-00*asterid II astV a rw I ~ Rosidae, Celastrales

arwH Rosidae, Rosales cRosidae, Cornales ham Id Asteridae, Campanulales Lag cpyc lAsteridae, Solanales FIGURE Astlad As e Asterales pa" eridae, cer 9~Asteridae, Calycerales c~~n Rosidae, Apiales guic Asteridae, Dipsacales of substitutions optimized onto one tree randomly selected from the 3,900 saved in Search II.) Genera marked with a "t" in A were omitted from Search II. Note that A is the consensus tree of Search I, whereas B is a single tree with branches not present in the strict consensus of Search II marked by an arrow. 578 Annals of the Missouri Botanical Garden

15A Nicotiana SolanaceaeC _ %Lycopersicon'etunia Convolvulus Convulvulaceae C Ipompea - Monfinia _ Grossulariaceae Brorgo Bo elo ium _ Boraginaceae Hydropl Hydrophylaceaec Ca iiche Callitrichaceae b Digitalis S]Schrophulariaceaef An*tihinumfNelsonia Nelsoniaceae Justicia americanat Jlusticia odora _ I R~ypoestes Acanthaceae f _Aphelandra AcanthusA Thunbergia Thunbergiaceaef Phaulopsist Ruellia Acanthaceaef Lepidagathis Sesamum Pedaliaceaef Verbena Verbenaceae Teucrium Lamiaceaeg Clerodendrum Verbenaceaeg Prostanthera Scutellaria lvia Lamiaceaeg Gkchomat tPhysostegwa Pogkostemon Caiiicarpa Verbenaceaeg r PHraozoasgc~s~e~ Pedaliaceae Buddleja Buddlejaceaef Catalpa Bignonlaceae _Steptocarpus Gesneriaceae _c inguculari Lentibulariaceae f _ Jasminum f Ligustrum Oleaceae Gentiana Gentianaceaee Apocynum Apocynaceaee Asckleias Asclepiadaceaee }_L... tpige ia Wet~emium Loganiaceaee Pentas jdacaRubiaceaed Iydrcleat Hydrophy aceae C _=Aucuba Cornacede b I Garrya Garryaceaeb * Eucommia Eucommiaceaea

aSth A asterid I ast IV Hamamelidae, Eucommiales jLG~rosS bRosidae, Cornales j~z ros~l cAsteridae, Solanales jham II dAsteridae, Rubiales ran eAsteridae, Gentianales lauau n fAsteridae, Schrophulariales ]| 1 ~~cmeP gAsteridae, Lamiales PM con .hAsteridae, Callitrichales cyc 'Rosidae, Rosales FIGURE15. The second clade of traditionalAsteridae. (Numbers above the branchesin B are the numbersof substitutionsoptimized onto one tree randomlyselected from the 3,900 saved in SearchII.) Generamarked with a "t" in A were omitted from Search II; genera marked with an asterisk in B were not available for Search I. Note that A is the consensus tree of Search I, whereas B is a single tree with branches not present in the strict consensus of Search II marked by an arrow. Volume 80, Number 3 Chase et al. 579 1993 Phylogenetics of Seed Plants

15B 11 Nicotiana 29 13o jSolanaceaei 13 20eL9AI:nea47icn 17 31 Convolvulus 1 omoea Convulvulaceae 8 Z5 Montinia Grossulariaceae e 22 2 Borago 1 2Heliotropm Boraginaceae g 10 Hydrophyllhjd i Eyiordicyl^on [ydrophyllaceae 8 Callitric e Callitrichaceae h 1 AnDigitanum Scrophulariaceae f 1710 Justicia

7 ~~13Bara1024 HyoestesLepidagathis Acanthaceaef 10 4 10 14 13Ruelliz 4 60 ~~1430 7AphelandzraAcanthus _ 12 Thunbergia Thunbergiaciae f 3 30Nelsonia Nelsoniaceae 10 2 Utricularia . f 20 _ 29 3Pinguicula Lentibulariaceae 28 Streptocarpus Gesneriaceae

26 Physostegia Lamiaceae g 2 8 9 Pogostemon 8,1 22 Clerodendron Verbenaceae g 24 Scutellaria 33Prostanthera Lamiaceae g 3 3 2 ~~~~~~~Salvia 2 ^3 CalVicarpa Verbenaceae g 12Verbena s 1 1! _ 2 _ 12Sesamum "Hroopyu Pedaliaceae 1l- a 10 Buddleja Buddlejaceae f 15 ~~44 Catalpa Bignon aceae 34Byblis* Byblidaceaee 7 _ 28 ~ siumsinuum- Oleaceae f 1 e 8 36 Vahlia* Saxifragaceae 11 14 Gentiana CGentianaceae C 7Pentas Rubiaceae d 16 31 7Apocynum Apocynaceae c 8 3 Asclefis Asclepiadaceaec 10Gelsemium ]Loganiaceae 1010 i2EAucuba[ Cornaceae b j?G~~('anrya Garryaceae a Eucommia Eucommiaceae

ast I ast IV asterid I V rosroast II aHamamelidae, Eucommiales rosIII b Rosidae, Cornales pil CAsteridae,Gentianales 1 ranl dAsteridae, Rubiales mag e Rosidae, Rosales lau pal I f Asteridae, Schrophulariales r 1 ~~~~cer strdeLmas rL Cpi~~~~n h!Asteridae,Callitrichales I cpyc I~~~~Asteridae,Solanales Nicotiana Solanaceae d3 _ - Eucommia A 4- 0 Aucuba iornaceae d5 Id4- d44 Garra Garryaceae I4 Rhododendron Ericaceae di T17 16 epa Hydrangeaceae

21 Paeonia Paeoniaceae 10 Ribes_Grossulariaceae d3 8 Heufhera Saxifragaceae

2 3 Cercidjphyllum bf dO 11 Altingth L d>5 10 Liquidambarorientalis 21 Rhodoleja 5 2 r Daphniphyllum dl dl Hamamelis 2 Sargentodoxa Sargentodoxaceae 17 ~Pisum I aaceae d2 d>5 17d Medicaflo Fab aceae 18 J10ygalaPlglca 8 6 LMyrca Ioyalaceae 4 11 2 Betula d4 d l>59 Casuarina dl dl 10 12 Cai|Dr 14 yagus C 1 rir21 Chojgus r d> 6 dSd5 T88 TLitnoeiaInus 6i 1> ofgus balansae d>5 d2 ~~~17L.-Nohofagus dombexi 10 12 Conarea oriasraceae d2 1 Datisca Datiscaceae 4 S c3 Luffa Cucurbitaceae d4 27 Be onna Begoniaceae d2 7 ~~~~~~16 Te rqmeles Daliscaceae

16 d4920 Trema 8I>5 12 Boehmeria D 121 3 Ficus

8 6 5 m17Prunus Rosaceae 2 5 17S Niraea 30 Bl1311i~celka. Malpi~rica cI>5 UmJ einra E ~~J 2 d~5 dih Bursera Burseraceae 7> 247 CahlhAcer Aceraceae 27 Schinus Anacardiaceae 1 Gunnera Gunneraceae d3 28 20 Akebi~~~yrsTrochodendulusza Mgharizaaaceae F 15 Eupteltron.etrac I H Cu 30~~ Pac~iysandra BNuxaceae 11 _ Sabica Sabiaceae 6 Lambertia Proteaceae polyphyleticHmeis iNelumbo Nelumbonaceae atwhchabrnh eomsa oytm28 winei Platanus a G consensusomspsmisicteaenceb"O22 Akebia Lardizabalaceae 7 Mahonia a Berbrxldaeat15 24 Caulophyllum ere esigateae dl dl 16 921 Ranunculus dl 9 20 dl 22Xanthorhiza Ranunculaceae d2 do ~ 19 Caitha 111 26 Cocculus Menispermaceae

15s 2 Euptelea ~ J 10 DietaFumariaceaeD 12 >5~P0 apaver Papaveraceae

FIGURE 16. An example of using the general analysis to focus a narrower study of internal support for a polyphyletic Hamamelidae. Numbers above the horizontal lines indicate the number of substitutions optimized (ACCT- RAN) onto one of the Most parsimonious trees found using character-state weighting (i.e., these are Fitch steps, equal