American Journal of Botany 96(1): 22–66. 2009.

R ECONSTRUCTING THE ANCESTRAL ANGIOSPERM FLOWER AND ITS INITIAL SPECIALIZATIONS 1

Peter K. Endress2,4 and James A. Doyle3

2 Institute of Systematic Botany, University of Zurich, Zollikerstrasse 107, 8008 Zurich, Switzerland; and 3 Department of Evolution and Ecology, University of California, Davis, California 95616 USA

Increasingly robust understanding of angiosperm phylogeny allows more secure reconstruction of the fl ower in the most recent common ancestor of extant angiosperms and its early evolution. The surprising emergence of several extant and fossil taxa with simple fl owers near the base of the angiosperms — Chloranthaceae, Ceratophyllum , Hydatellaceae, and the Early Cretaceous fossil Archaefructus (the last three are water plants)— has brought a new twist to this problem. We evaluate early fl oral evolution in an- giosperms by parsimony optimization of morphological characters on phylogenetic trees derived from morphological and molecu- lar data. Our analyses imply that Ceratophyllum may be related to Chloranthaceae, and Archaefructus to either Hydatellaceae or Ceratophyllum. Inferred ancestral features include more than two whorls (or series) of tepals and stamens, stamens with protrud- ing adaxial or lateral pollen sacs, several free, ascidiate carpels closed by secretion, extended stigma, extragynoecial compitum, and one or several ventral pendent ovule(s). The ancestral state in other characters is equivocal: e.g., bisexual vs. unisexual fl owers, whorled vs. spiral fl oral phyllotaxis, presence vs. absence of tepal differentiation, anatropous vs. orthotropous ovules. Our results indicate that the simple fl owers of the newly recognized basal groups are reduced rather than primitively simple.

Key words: ancestral fl owers; angiosperm phylogeny; ANITA grade; Archaefructus ; basal angiosperms; Ceratophyllum ; Chloranthaceae; fl ower evolution; Hydatellaceae; water plants.

The question of the structure and biology of the ancestral lecular results, have linked glossopterids, Pentoxylon , Bennet- angiosperms, and especially their fl owers, is an enduring riddle. titales, and Caytonia , with or without Gnetales, with angiosperms Although we are continually gaining new insights from new ( Bateman et al., 2006 ; Doyle, 2006 ; Friis et al., 2007 ; Frohlich fossils and new studies on phylogeny, morphology, and devel- and Chase, 2007), but there is no general agreement that any of opmental genetics in extant plants, we are still far from a fi nal these taxa are related to angiosperms. The question of still answer. There are gaps at different levels. First is the uncer- closer angiosperm stem relatives is still a void because there are tainty concerning which other seed plants are the closest rela- no fossils that undisputedly represent this part of the tree. tives of angiosperms, particularly extinct groups because most Fortunately, there has been much more progress in recon- molecular analyses indicate that no living group of gymno- struction of the fi rst crown group angiosperms. Recent work on sperms is any closer to angiosperms than any other. Second, early fossil angiosperms (reviewed by Doyle, 2001, and Friis even if known fossils can be recognized as angiosperm stem et al., 2006 ) and on extant “ ANITA grade ” angiosperms relatives, all such groups are morphologically well removed ( Endress, 2001 , 2008a) has provided new insights. Problems at from angiosperms, so there is still a major gap that can only be this level have become easier to tackle thanks to analyses of liv- fi lled by the discovery of closer stem relatives. Third is the ing angiosperms, particularly using molecular data, which have problem of the original morphology and early evolutionary dif- clarifi ed relationships within the crown group with a degree of ferentiation of crown group angiosperms. precision and statistical confi dence barely imaginable two de- Identifi cation of seed plant relatives of the angiosperms has cades ago. These analyses have consistently rooted the an- been one of the most contentious issues in plant systematics and giosperm phylogenetic tree among the ANITA lines, namely evolution, both before and after the introduction of phyloge- Amborella , Nymphaeales, and Austrobaileyales ( Mathews and netic methods ( Crane, 1985 ; Doyle and Donoghue, 1986 ; Nixon Donoghue, 1999 ; Parkinson et al., 1999 ; Qiu et al., 1999 ; Renner, et al., 1994; Doyle, 1994, 1996 ). Molecular analyses contradict 1999 ; Soltis et al., 1999, 2000 ; Barkman et al., 2000 ; Graham one of the few points on which morphological analyses agreed, and Olmstead, 2000; Zanis et al., 2002), which has focused at- that Gnetales are the closest living relatives of angiosperms tention on these taxa as particularly likely to yield insights on (Donoghue and Doyle, 2000; Burleigh and Mathews, 2004; the fi rst angiosperms (Doyle and Endress, 2000; Endress and Soltis et al., 2005 ), but they say nothing about fossil relatives. Igersheim, 2000a, b ; Endress, 2001, 2004 , 2006, 2008a; Fried- Several recent studies, some of which take into account mo- man and Williams, 2003 , 2004 ; Williams and Friedman, 2004 ; Friedman, 2006; Endress and Doyle, 2007). The main uncer- tainty is whether Amborella and Nymphaeales form two succes- 1 Manuscript received 7 February 2008; revision accepted 12 September 2008. sive branches or a clade (Barkman et al., 2000), with some The authors thank E. M. Friis and M. Frohlich for useful discussions and recent support for the latter hypothesis from mitochondrial genes suggestions that improved the manuscript. J.A.D. thanks P. Garnock-Jones (Qiu et al., 2006), but the former supported by recent analyses of and the School of Biological Sciences, Victoria University of Wellington, entire plastid genomes (Jansen et al., 2007; Moore et al., 2007). for facilities and a supportive environment during preparation of this paper. This work was facilitated by travel support from the NSF Deep Time An alternative rooting based on plastid genomes of fewer taxa, Research Coordination Network (RCN0090283). with grasses the sister group of all other angiosperms ( Goremykin 4 Author for correspondence (e-mail: [email protected]) et al., 2003), appears to be an artifact of low taxon sampling and long branch attraction ( Degtjareva et al., 2004 ; Soltis and Soltis, doi:10.3732/ajb.0800047 2004 ; Stefanovic et al., 2004 ; Leebens-Mack et al., 2005 ). 22 January 2009] Endress and Doyle — Ancestral flowers 23

All other angiosperms form a strongly supported clade, named could be said to be typical at a relatively “ basal ” level of angio- Mesangiospermae by Cantino et al. (2007) , but relationships sperms (groups other than monocots and eudicots, or “ Magno- among several lines in this clade remain poorly resolved, prob- liidae” in the paraphyletic sense of Takhtajan, 1964), but ably as a result of very rapid radiation (Moore et al., 2007). One because they were scattered in different taxa (e.g., anther open- important area of current uncertainty is the position of Chloran- ing by valves, spiral fl oral phyllotaxis, inner staminodes, trim- thaceae, which have been the subject of much discussion be- erous fl owers), it was possible to entertain several alternative cause of their extremely simple fl owers. Combined analyses of models for the ancestral fl ower (e.g., Endress, 1986a ). However, morphological and molecular data (Doyle and Endress, 2000) especially since 1999, a more precise discussion is possible be- and some molecular studies ( Qiu et al., 2005 ; Duvall et al., cause phylogenetic reconstructions are generally more advanced, 2006 ; Mathews, 2006 ) have placed Chloranthaceae at the base and specifi cally the topology of the basal grade of extant angio- of mesangiosperms, but they are nested within mesangiosperms sperms is well supported and can be used as a basis for discus- in most molecular trees, including most of those found in analy- sions on evolution. As emphasized by Crisp and Cook (2005), ses of complete plastid genomes (Jansen et al., 2007; Moore it cannot be assumed that single low-diversity “ basal ” lines are et al., 2007). Suggestions that fl owers of Chloranthaceae were plesiomorphic in any given character, but when several lines primitive based on the abundance of apparently related fossils branch sequentially below the vast bulk of a clade, as is appar- in the Early Cretaceous (reviewed by Eklund et al., 2004 ; Friis ently the case for angiosperms, and these lines share the same et al., 2006 ) have faded with fi rm establishment of the basal character state, this state can be reconstructed by parsimony ANITA grade, but if Chloranthaceae are sister to the remaining analysis as ancestral. We took advantage of the new evidence mesangiosperms they could still be relevant to reconstruction on rooting in an analysis of basal angiosperms (including basal of the original fl ower and its initial modifi cations. monocots and basal eudicots), in which we used parsimony op- Comparative studies of fl oral developmental genetics repre- timization on a tree based on morphological data and rbcL , sent another growing fi eld that promises to provide new insights atpB, and 18S rDNA sequences (Soltis et al., 2000) to estimate on early fl oral evolution. Such studies have already been used for ancestral states and trace character evolution ( Doyle and interpolations between angiosperms and other living seed plants Endress, 2000). In later articles we concentrated on implica- and within angiosperms ( Frohlich and Parker, 2000 ; Frohlich, tions of this data set for evolution of pollen morphology (Doyle, 2003 , 2006 ; Baum and Hileman, 2006 ; Irish, 2006 ; Soltis et al., 2005 ), leaf architecture ( Doyle, 2007 ), fl oral phyllotaxis ( Endress 2006 ; Frohlich and Chase, 2007 ; Theissen and Melzer, 2007 ). and Doyle, 2007 ), and the position of Hydatellaceae ( Saarela Several spectacular new fi ndings have brought the aquatic et al., 2007). This “ angiosperm-centered ” or “ top-down ” approach habitat to the center of the attention and debate on early angio- (Bateman et al., 2006) can be questioned on the grounds that in sperm evolution (e.g., Sun et al., 2002; Friis et al., 2003; Crepet theory outgroup and ingroup relationships cannot be addressed et al., 2004 ; Feild and Arens, 2007 ). These are (1) the recogni- separately. However, in practice this seems less problematical tion of new aquatic angiosperms in the Early Cretaceous fossil than anticipated, thanks to the increasingly robust rooting of record, including Archaefructus ( Sun et al., 1998 , 2002 ; Friis angiosperms based on molecular data. et al., 2003 ; Ji et al., 2004 ), which had fertile axes bearing paired In the present paper we discuss changes in our perception of stamens, single or paired carpels, and no perianth; Monsechia , the fl ower in the most recent common ancestor of living angio- tentatively interpreted as a bryophyte when it was fi rst described sperms (the crown group node) and its initial evolutionary mod- (Gomez et al., 2006); and Scutifolium , assigned to Cabom- ifi cations, using an updated version of the Doyle and Endress baceae ( Taylor et al., 2008 ), in addition to previously recog- (2000) data set and taking into account new evidence from phy- nized water plants such as Nelumbites (Doyle and Hickey, logenetic and structural studies on extant plants, fossils, and 1976 ; Upchurch et al., 1994 ; Mohr and Friis, 2000 ; Wang and evo-devo studies. It is unlikely that this “ ancestral fl ower ” was Dilcher, 2006 ); (2) the discovery that the submerged water plant the “ fi rst fl ower” in a morphological sense, which may have family Hydatellaceae belongs not in monocots but rather to originated much earlier on the angiosperm stem lineage. We Nymphaeales in the ANITA grade ( Saarela et al., 2007 ); and will not consider the origin of the angiosperm fl ower in relation (3) indications from some (though not most) molecular analy- to reproductive structures in outgroups, which requires consid- ses that the aquatic genus Ceratophyllum (= Ceratophyllaceae), eration of fossil seed plants, a topic treated elsewhere ( Doyle, which had a brief period of fame as the inferred sister group of 2006 , 2008). all other angiosperms in analyses of rbcL (Chase et al., 1993), Our previous study (Doyle and Endress, 2000) presented a may belong just above the basal angiosperm grade, with Chlo- list of inferred ancestral states for all characters, but this needs ranthaceae ( Duvall et al., 2006 ; Mathews, 2006 ; Qiu et al., reassessment in light of new data. Since 2000, we have been 2006). The importance of fresh-water habitats and fl ood plains revising our data set by adding new characters, refi ning old ones, in early angiosperm history has long been recognized by paleo- adding new taxa, and splitting taxa into more homogeneous botanists (Doyle and Hickey, 1976; Taylor and Hickey, 1992), units to analyze character evolution in more detail and fi ll in and continues to be a major topic of discussion by paleoecolo- less well sampled parts of the tree. We have not yet performed gists ( Mart í n-Closas, 2003 ; Feild et al., 2004 ; Coiffard et al., a new combined analysis of morphological and molecular data, 2007; Feild and Arens, 2007). The impression that Early Creta- but we have made changes in the tree used as a framework for ceous angiosperms included a large number of water plants discussion where recent data provide robust evidence for differ- may be partly due to a bias in favor of fossilization of aquatics ent relationships. For example, our previous combined analysis over other plants, but water plants were clearly more common linked Piperales with monocots, but accumulating molecular data than expected under the old view that the initial diversifi cation (Zanis et al., 2002; Sauquet et al., 2003; Qiu et al., 2005, 2006 ) of angiosperms involved woody plants (cf. Doyle and Hickey, consistently associate them with Canellales (Canellaceae, Win- 1976 ). teraceae), Magnoliales, and Laurales, in a clade named Magno lii- Until about ten years ago, only a vague recognition of more dae by Cantino et al. (2007) , not to be confused with Magnoliidae widespread features in basal angiosperms was possible. They in the paraphyletic sense of Takhtajan (1964) and others. 24 American Journal of Botany [Vol. 96

Our most important change in taxon sampling is the addition phylogenetic analyses, particularly those related to rooting, to estimate ances- of two aquatic groups: Hydatellaceae, now linked with Nympha- tral states. Our assumptions on rooting of taxa and the publications on which eales ( Saarela et al., 2007 ), and Ceratophyllum . In Doyle and they are based are cited in the taxon list. Improved information on relationships within taxa has led us to make many minor changes in scoring of taxa since Endress (2000), we omitted Ceratophyllum because many char- Doyle and Endress (2000) . acters in our matrix were lacking or uninterpretable due to re- duction, its position was unstable in preliminary analyses, and Taxa— Besides adding Ceratophyllum and Hydatellaceae, we have in- we assumed it would have a minor effect on inferences on char- creased our taxon sampling in other groups. In Chloranthaceae, we split Chlo- acter evolution because of its specialized, reduced nature. How- ranthus and Sarcandra , treated as one taxon in Doyle and Endress (2000), into ever, omitting Ceratophyllum is no longer justifi able in light of the two genera and rescored several characters based on Eklund et al. (2004). In the increasing number of other near-basal taxa with simple Ranunculales, we have added Circaeaster and split Hydrastis and Glaucidium from “ core ” Ranunculaceae (their sister group according to Hoot et al., 1999) fl owers and the suggestion that they represent a prefl oral state because they differ substantially in fl oral features and may thus affect recon- ( Friis and Crane, 2007 ). Whether such fl owers are reduced struction of fl oral evolution. For the same reason, we split Magnoliaceae into should be tested rather than assumed. The claim that Cerato- Liriodendron and Magnolioideae ( Magnolia s. l. of recent authors) and Trocho- phyllum is the sister group of eudicots, based on analyses of dendraceae into Trochodendron and Tetracentron . We have modifi ed the scor- complete plastid genomes ( Jansen et al., 2007 ; Moore et al., ing of Platanaceae (now Platanus ) and Buxaceae, in which we previously 2007), also needs to be evaluated in light of morphological data included presumed fossil relatives, to apply strictly to the extant crown groups, in anticipation of testing the position of the fossils. We have increased our sam- and its implications explored. pling of Alismatales for future tests of comparisons with fossils. We have added Another major change is addition of the Early Cretaceous Nartheciaceae because they appear to be related to but less modifi ed than Di- fossil plant Archaefructus , which a cladistic analysis by Sun oscoreaceae ( Caddick et al., 2002a ), and Melanthiaceae as a relatively ple- et al. (2002) identifi ed as the sister group of all extant angio- siomorphic exemplar of Liliales ( Chase et al., 2006 ). sperms (i.e., a stem relative). This interpretation was questioned In Ceratophyllum, the fertile structures have been variously interpreted by Friis et al. (2003), who interpreted Archaefructus as a crown ( Endress, 1994b ; Iwamoto et al., 2003 ): at one extreme, as female fl owers with a single carpel surrounded by tepals and as male fl owers with tepals and numer- group angiosperm with reduced unisexual fl owers, but reaffi rmed ous stamens; at the other, as female fl owers with no perianth but bracts lower by Crepet et al. (2004). In either case, its unusual combination on the axis and as spikes with basal bracts and numerous male fl owers consist- of characters make it potentially relevant to reconstruction of ing of one stamen, with no perianth or individual subtending bract (Endress, the ancestral fl ower. The addition of Archaefructus is part of a 2004). For the purposes of this analysis, we have provisionally accepted the general effort to integrate fossils into the phylogeny of living second interpretation. First, in the female structures, single carpels occasionally basal angiosperms (Doyle and Endress, 2007). Results of our occur in the axils of the sterile appendages ( Aboy, 1936 ; Iwamoto et al., 2003 ), suggesting that the latter are bracts rather than tepals and the system is a re- analyses concerning other fossil taxa are not presented here be- duced infl orescence. Second, the stamens have an extremely labile phyllotaxis cause they have less impact on reconstruction of the ancestral and marked acropetal delay in maturation (Endress, 1994b), which is a common fl ower. Some of these fossils are too deeply nested within mag- pattern in fl owers of spicate infl orescences but anomalous within the androe- noliids and eudicots to affect inferred ancestral states; others cium of a multistaminate fl ower. may be more basal (e.g., taxa apparently related to Chloran- Our scoring of Archaefructus is based on whole plants of A. liaoningensis thaceae: Eklund et al. [2004]) but add few new elements be- and A. sinensis (Sun et al., 1998, 2001 , 2002 ; Friis et al., 2003); features are generally consistent in A. eofl ora ( Ji et al., 2004 ), which may represent either a cause they resemble their presumed extant relatives in fl oral smaller species or a younger stage of A. sinensis . We analyzed the position of features. Archaefructus using two alternative scorings, one (Archaefructus inf) assuming Besides considering implications of current phylogenies for that fertile axis was a raceme of male and female fl owers consisting of usually evolution of individual fl oral characters and character com- two stamens and one or two carpels, the other (Archaefructus fl o), following Sun plexes, we stress several specifi c broader issues. These include et al. (2002) , that it was a bisexual fl ower or prefl ower with paired stamens be- (1) what present data say about evolutionary interpretation of low and carpels above. Friis et al. (2003) questioned whether the bodies that Sun et al. (2001, 2002 ) described as pollen were in fact pollen grains, because of their the fl owers of Magnoliales and Winteraceae, once widely as- irregular size and shape, but we provisionally assume that at least some of them sumed to be primitive; (2) whether the simple fl ower structure are pollen and have scored them based on the most convincing specimen, illus- in some basal angiosperms (Hydatellaceae, Archaefructus , Cer- trated in Fig. 2F of Sun et al. (2002) . To evaluate the alternative interpretation, we atophyllum, Chloranthaceae) is due to reduction of more “ com- have used a third scoring ( Archaefructus NP) that corresponds to Archaefructus plete ” fl owers, retention of a “ prefl oral ” state, or to breakdown inf with pollen characters treated as unknown. Sun et al. (1998, 2002 ) described of the distinction between fl owers and infl orescences due to the carpels as conduplicate (= plicate), but in extant carpels of similar appear- ance this cannot be determined without developmental or anatomical evidence loss of fl oral identity, issues raised by Friis and Crane (2007) (Friis et al., 2003; Endress, 2005). They described the fruits as follicles, but they and Rudall et al. (2007), and if and how this might be related to did not actually report dehiscence. The seeds appear to have a palisade exotesta an aquatic habit; and (3) what the evolutionary consequences of as defi ned here (character 101, including not only radially elongated but also a position of Chloranthaceae just above the basal grade, with or shorter sclerotic cells): Sun et al. (1998 , 2002 ) described the surface as consist- without Ceratophyllum ( Doyle and Endress, 2000 ; Duvall ing of epidermal cells with cutinized anticlinal and periclinal walls. et al., 2006 ; Mathews, 2006 ; Qiu et al., 2006 ), would be for in- Ji et al. (2004) interpreted seeds of A. eofl ora as orthotropous, but their pub- lished illustrations are not clear enough to determine whether the seeds were terpretation of the fl owers in these groups, and how these would anatropous or orthotropous; the fi gure of the end of a seed in Fig. 2C of Sun differ in the context of the plastid genome trees ( Jansen et al., et al. (1998) is actually more suggestive of an anatropous ovule. Hence we have 2007 ; Moore et al., 2007 ), where the two taxa are nested sepa- scored ovule curvature (93) as unknown. Ji et al. (2004) described one lateral rately within mesangiosperms. unit in A. eofl ora as bisexual, with one stamen and two carpels, and they also interpreted one unit in the type specimen of A. sinensis ( Sun et al., 2002 ) as bisexual. Under our character defi nition, A. eofl ora might be scored as uncertain (0/1) for fl ower sexuality (26). However, we believe it would be premature to MATERIALS AND METHODS rescore Archaefructus as a whole in this way.

Lists of taxa and characters and the data matrix are presented in Appendix 1. Characters— In this study we have not included all the characters in our In dealing with characters that vary within taxa, we have not simply scored most recent version of the Doyle and Endress (2000) data set, many of which characters as uncertain ( “ polymorphic ” ) but have made use of results of are not relevant for our present purposes, where we have used fi xed backbone January 2009] Endress and Doyle — Ancestral flowers 25

constraint trees as a framework for placement of Ceratophyllum and Archae- Scoring the number of perianth and stamen whorls (34, 43) is straightfor- fructus and reconstruction of fl oral evolution, and would require excessively ward in whorled taxa (except for seemingly tetramerous fl owers that actually lengthy documentation and argumentation. We have included all fl oral charac- have dimerous whorls, as in Proteaceae, Tetracentron , and Buxaceae, as in- ters, including those of stamen, carpel, and ovule morphology. In addition, we ferred from the fact that the stamens appear to be opposite the tepals: von Balt- include all those nonfl oral characters needed for analysis of the position of Cer- hazar and Endress, 2002a; Chen et al., 2007 ), but again the treatment of spiral atophyllum and Archaefructus . Characters omitted because they do not exist or taxa poses problems. Many spiral taxa (and Nelumbo , with chaotic stamen in- are inapplicable in Ceratophyllum include aspects of secondary xylem and sertion on an androecial ring meristem: Hayes et al., 2000) have numbers of phloem, leaf anatomy, and the inner and outer integuments, which are reduced tepals and/or stamens that are comparable to those of taxa with more than two or fused into a single integument. whorls, so we have scored them accordingly. We also used the number of series Some of the most important and complex arguments for decisions in defi ni- (in which a certain number of parts fi lls the circumference of the fl ower; En- tion of characters and scoring of particular taxa are discussed in this section, dress and Doyle, 2007 ) as a rough substitute for number of whorls. others that are less problematic or signifi cant in Appendix 1. Because of space In most basal angiosperms, all perianth parts are best described as tepals limitations, we cite only general sources of information for particular character ( Hiepko, 1965 ; Walker and Walker, 1984 ; Endress, 2001 ; Ronse De Craene, sets and especially important references on particular taxa, and reserve more 2008 ) because they are less strongly differentiated than the typical sepals and detailed documentation and resolution of differences between our interpreta- petals of core eudicots ( Pentapetalae of Cantino et al., 2007). These tepals may tions and those of other workers for elsewhere. We concentrate on references be uniform (either sepaloid or petaloid) or differentiated into outer sepaloid and for new taxa and characters; for those used in Doyle and Endress (2000), read- inner petaloid parts, distinctions recognized in character 35. We include both ers are referred to that article. tepals and more differentiated petals in the count of whorls, and staminodes as Our general philosophy on defi nition of characters is explained in more de- well as fertile stamens. However, we also introduced a separate character (36) tail in Doyle and Endress (2000) . Few of our characters are quantitative in the for presence or absence of typical petals (mostly in Ranunculales), defi ned on sense of continuous (e.g., pollen size, nexine thickness), but there are often se- more pronounced differences in anatomy and delay in development. Taxa with ries of conditions that could be grouped into many states or a few. In general, petals may show differentiation within the outer perianth whorls, such as we have tried to break the variation into a smaller number of states in ways that Nuphar , which has outer sepaloid and inner petaloid “ tepals ” or “ sepals ” and make morphological (especially developmental) sense and reduce the number much smaller petals. of uncertain ( “ polymorphic ” ) scorings of taxa (assuming that this reduction is We have made fewer changes from Doyle and Endress (2000) in characters evidence that the variations included in each of the states are related). Several of individual fl oral parts. Following Eklund et al. (2004) , to reduce uncertain important changes concern replacement of multistate with binary characters, scorings, we modifi ed the stamen base character (48) to combine short and wide which can sometimes improve resolution of relationships in cases where the and short and narrow in the same state, and the orientation character (53) to optimization of a multistate character would be ambiguous. In several cases we combine slightly introrse with latrorse. We previously treated modes of carpel previously used unordered multistate characters to combat the Maddison “ long sealing as a multistate character (corresponding to the four types of Endress and distance ” effect ( Maddison, 1993 ): where the ancestral state in one clade in Igersheim, 2000a). However, carpel sealing has two potentially independent which a structure (more generally a character) occurs in two (or more) versions aspects, degree of postgenital fusion and secretion, which we have split into infl uences the polarity of the character in another clade that has the structure, two characters (76, 77). We separated types of papillae (82) from larger protu- even though the structure does not exist in the intervening lines and presumably berances (81), because pluricellular papillae and protuberances co-occur in Am- arose independently. This artifact can be avoided by treating lack of the struc- borella and Trimenia but not in other taxa and therefore appear to represent ture as one state of a multistate character and different versions of the structure independent characters. as other states. However, this procedure weakens the contrast between presence Many aspects of fl oral evolution were also treated by Ronse De Craene et al. and absence of the structure as an independent source of information on (2003) . They made less effort to ensure independence of characters: for exam- relationships. ple, lack of perianth was a state in three of their characters. This was not neces- An example that underlines the importance of the Maddison effect concerns sarily a problem in their study, in which they plotted characters on a molecular presence or absence of a perianth. In Doyle and Endress (2000), we treated the tree, and it may be useful in assessing the implications of different character number of perianth whorls as an unordered multistate character, with no peri- defi nitions. However, such redundancy poses problems if characters are used anth one of four states. A group where this may cause problems is Chloran- for tree reconstruction, since it may overweight what was presumably a single thaceae, where Hedyosmum has one perianth whorl and the other genera have change— for example, loss of perianth. Because we intend to use our data set in no perianth. Since most outgroups have a perianth, its presence might appear to a future combined analysis and have used it to investigate the relationships of be evidence for the basal position of Hedyosmum , or in other words its loss Ceratophyllum and Archaefructus in the current study, we have tried to mini- could be evidence for the monophyly of the remaining genera (which is sup- mize redundancy among characters. ported by molecular evidence). However, when presence and number of whorls Infl orescence characters deserve special attention as an area where we have are treated as a single unordered multistate character, scoring Hedyosmum as made major modifi cations. In our previous analysis ( Doyle and Endress, 2000 ), having one whorl does not favor a basal position in Chloranthaceae because the we recognized a relatively crude infl orescence character emphasizing degree of outgroups have two or more whorls, states that are not recognized as any more branching, with three states: solitary fl owers; racemes, spikes, and botryoids; similar to one whorl than to none. For this reason, Eklund et al. (2004) split the and more richly branched infl orescences such as panicles and compound infl o- Doyle and Endress (2000) character into two— one for presence or absence of a rescences of racemes, spikes, and botryoids. Thus in infl orescences of the sec- perianth, the other for number of whorls, with taxa lacking a perianth scored ond state, we did not recognize the standard contrast between indeterminate and as unknown — and we have followed this solution here (characters 31, 34). determinate infl orescences, or the related distinction of Troll (1964) and We- Maddison (1993) pointed out cases where this procedure is unlikely to cause berling (1989) between polytelic systems with no terminal fl ower (racemes, problems, notably where loss of a structure occurs in a terminal clade. With spikes, thyrses) and monotelic systems in which all axes terminate with a fl ower relationships largely inferred from molecular data, cases where this may cause (botryoids, thyrsoids, panicles— all sometimes imprecisely described as artifacts can usually be recognized and treated in discussion. “ cymes ” ). This was because of a perception that the two types intergrade within Additional important changes involve other characters of fl oral organization. taxa, such that many taxa would have to be scored as uncertain. However, In Doyle and Endress (2000), we recognized phyllotaxis and merism (merosity) closer examination has led us to conclude that a different grouping of traditional as separate characters in both the perianth and the androecium, but this poses types into basically monotelic and polytelic states (in character 22, Fig. 1 ) leads problems for scoring of merism in spiral taxa. Our solution was to treat merism to fewer problems than we had thought and is more informative: one state in- as a multistate character, with spiral taxa scored as (0) irregular and whorled cludes units lacking a terminal fl ower (racemes, spikes, thyrses), the other those taxa as (1) trimerous or (2) dimerous, tetramerous, or pentamerous. However, with a terminal fl ower (botryoids, thyrsoids, panicles). Although taxa often this may introduce bias due to redundancy of spiral phyllotaxis and irregular vary between types within each of these states, there is less variation between merism. One solution would be to combine phyllotaxis and merism into a single types belonging to the different states. character, but as discussed in Endress and Doyle (2007) , the distinction between Racemes and thyrses differ on whether the lateral units on the indeterminate spiral and whorled appears to be consistent and independent enough to be axis are single fl owers or cymes. Cymes are branching systems that can have treated separately. Our solution is to retain both characters (32, 33 for the peri- one to several branching orders (i.e., a main axis and lateral branches of one to anth; 41, 42 for the androecium) but score spiral taxa as unknown for merism. several orders formed by repeated branching of these laterals), but each axis has Optimization of this character across the tree produces artifactual reconstruc- not more than two lateral branches of the next higher order, and all axes are tions of merism in spiral taxa, but this can be considered in discussion. usually determinate. Botryoids are the other way around: they have only one 26 American Journal of Botany [Vol. 96

state (in character 22, Fig. 1): there is variation between these two extremes in many taxa, such as Austrobaileya , Eupomatia , Magnoliaceae, and Calycan- thaceae. If the axillary branch (pedicel) bearing the fl ower has no appendages or at most one or two prophylls, we call the system a raceme; if it has more sterile appendages, we call the fl ower solitary. This defi nition allows most taxa to be scored unambiguously. Schisandraceae are still mixed (0/1), since the number of bracteoles varies between zero and three or more among species ( Weberling, 1988 ; Saunders, 2000 ). Special problems in interpretation of Nymphaeales are treated in the Discussion, since they make more sense in a phylogenetic context. When fl owers are unisexual and the infl orescences of male and female fl ow- ers differ in type, we have scored the taxon based on the more complex type. Thus, we have scored Hedyosmum , with male spikes and female thyrses, as having thyrses; and Ceratophyllum , with solitary female fl owers and male spikes, as having spikes. Except for three pollen characters (see Appendix 1), all multistate characters were treated as unordered.

Analyses— Our analyses (all based on parsimony) used “ backbone con- straint” trees, with Recent taxa fi xed into one of two topologies. Analyses were performed with the program PAUP* version 3.1.1 ( Swofford, 1990 ) and in- volved 10 or 100 heuristic replicates, stepwise random addition of taxa, and tree-bisection-reconnection (TBR) branch swapping. The relative parsimony of alternative relationships was determined by searching for trees less than or equal to a given number of steps and observing the trees obtained or by moving taxa manually with MacClade ( Maddison and Maddison, 2003 ). The fi rst backbone tree (henceforth labeled D& E) is a modifi cation of the tree found in our morphological and three-gene analysis ( Doyle and Endress, 2000), with changes where accumulating molecular data have most strongly and consistently contradicted relationships found in our previous study. Essen- tially this is a handmade supertree. Besides linking Piperales with Canellales, as already discussed, we have moved Euptelea from within Ranunculales to the base of the order, following Kim et al. (2004a) ; this position is actually more parsimonious in terms of morphology. Taxa added or split for the reasons dis- cussed earlier have been placed following Les et al. (1997) , Hoot et al. (1999) , Fig. 1. Sketches illustrating infl orescence types included in three Soltis et al. (2000) , Chen et al. (2004) , and Chase et al. (2006) . In a preliminary states of infl orescence character (22). (A, B) = state 0; (C – E) = state 1; analysis, we added Ceratophyllum to the data set and constrained all other rela- (F – H) = state 2. (A) solitary, terminal; (B) solitary, axillary; (C) botryoid; tionships as described. The tree found in this constrained analysis is the modi- (D) panicle; (E) thyrsoid; (F, G) raceme; (H) thyrse. fi ed D & E backbone tree used in subsequent analyses. The second backbone tree (labeled J/M) incorporates relationships of major clades found in analyses of whole plastid genomes by Jansen et al. (2007) and branching order (i.e., a main branch and lateral branches of only the fi rst order), Moore et al. (2007), notably with Chloranthaceae linked with magnoliids, Cer- but the number of fi rst-order lateral branches is not limited until the terminal atophyllum with eudicots, and the latter two with monocots. The same relation- fl ower is formed; as in cymes the axes are determinate. When there is only one ships were found by Saarela et al. (2007) in analyses of a smaller plastid data branching order and axes have only one or two lateral fl owers, cymes and set. Relationships within clades (which were sparsely sampled in the plastid botryoids cannot be distinguished unless more highly branched units are found. studies) are the same as in the D & E backbone tree. Racemes and thyrses both occur within taxa such as Chloranthaceae, where To investigate the position of Archaefructus, we analyzed the data set with Hedyosmum has thyrses of female fl owers and spikes of male fl owers, and some Archaefructus added, using both backbone trees. To assess implications of the species of Ascarina have spikes, others thyrses. We have recognized this dis- hypothesis that Amborella and Nymphaeales form a clade, we rerooted trees tinction with a separate character (23, lateral units single fl owers or cymes). We manually with the program MacClade version 4.03 ( Maddison and Maddison, have also distinguished racemes from spikes by introducing a character con- 2003 ). trasting pedicellate and sessile fl owers (24). We used MacClade to optimize character evolution on trees, reconstruct Another important distinction concerns the presence or absence of bracts or ancestral states, and identify characters supporting relationships. When we re- leaves (pherophylls) subtending the fl owers (25). In Archaefructus , Sun et al. fer to features as unequivocal synapomorphies of particular clades, this does not (2002) cited the absence of bracts below the paired stamens and carpels as evi- mean they are uniquely derived, but rather that the change in state unequivo- dence that the fertile axis was a fl ower (or prefl ower) rather than an infl ores- cally occurs at this point on the tree, as opposed to cases where the position of cence. However, subtending bracts are absent in several groups in the present change is equivocal (e.g., where an earlier origin followed by a reversal and two data set, such as Hydatellaceae, Acorus , and Araceae. later origins are equally parsimonious, or where the character state in neighbor- Other problems concern the distinction between solitary fl owers and ra- ing taxa is unknown). cemes, specifi cally when solitary fl owers are borne in the axils of more or less unmodifi ed vegetative leaves. Solitary axillary fl owers are sometimes distin- guished from lateral fl owers in a raceme based on whether they are subtended RESULTS by normal leaves or modifi ed bracts, but this is more a matter of degree than a fundamental difference in organization. This problem is illustrated by cases in which fl owers are borne in the axils of bracts on an axis that then reverts to When Ceratophyllum is added to the updated Doyle and En- producing vegetative leaves (e.g., Schisandra , Euptelea ; Endress, 1969 ; We- dress (2000) tree, its most parsimonious position is as the sister berling, 1988). In our previous analysis we scored these as solitary. Alterna- group of Chloranthaceae (776 steps; Fig. 2A ). It is nested within tively, even systems with fl owers in the axils of normal leaves are sometimes Chloranthaceae in all six trees that are one to three steps longer. described as racemes (Weberling, 1989). Because mode of branching seems A position as the sister group of eudicots (Jansen et al., 2007; more fundamental than variation between bracts and leaves, we have adopted this approach, grouping systems where fl owers are borne in the axils of bracts Moore et al., 2007 ) is nine steps less parsimonious (785 steps); and regular leaves as racemes. We group fl owers that terminate either a long a position as the sister group of monocots is eight steps less shoot (the classic terminal condition) or an axillary short shoot in the solitary parsimonious (784 steps). January 2009] Endress and Doyle — Ancestral flowers 27

Fig. 2. Representative most parsimonious trees obtained after addition of Archaefructus to backbone constraint trees of Recent basal angiosperms. OM and OE indicate presumed positions of other monocots and other eudicots, respectively. (A) Using D& E backbone tree, from combined morphological and molecular analysis of Doyle and Endress (2000) , with modifi cations based on more recent data. (B) Using J/M backbone tree, with relationships among major clades found in plastid genome analyses of Jansen et al. (2007) and Moore et al. (2007), but with relationships within clades as in Fig. 2A. Nymph = Nymphaeales, Aust = Austrobaileyales, Chlor = Chloranthaceae, Piper = Piperales, Ca = Canellales, Magnol = Magnoliales. 28 American Journal of Botany [Vol. 96

When Archaefructus is scored as having an infl orescence of supported by loss of bracts subtending the male fl owers (25) unisexual fl owers (Archaefructus inf) and added to the D & E and dry fruit wall (97). backbone tree, its single most parsimonious position is as the A sister group relationship of Ceratophyllum and eudicots, as sister group of Hydatellaceae (782 steps; Fig. 2A). Its next best found in the plastid genome analyses of Jansen et al. (2007) and position (one step worse) is sister to the remaining Nymphae- Moore et al. (2007) and many other molecular analyses (e.g., ales (henceforth designated “ core Nymphaeales” ). Seven posi- Saarela et al., 2007), is nine steps less parsimonious and would tions are two steps worse: sister to all Nymphaeales, Cabomba , be supported by only one unequivocal morphological synapo- Ceratophyllum, the Chloranthaceae- Ceratophyllum clade, all morphy, dry fruit wall (97), a highly homoplastic character. It is mesangiosperms except the Chloranthaceae-Ceratophyllum eight steps less parsimonious to link Ceratophyllum with mono- clade, and either Euptelea or Circaeaster in the eudicots. cots, which would be supported by loss of cambium (4). The J/M backbone tree based on plastid genome data (Jansen Whether this parsimony differential is suffi cient to overrule the et al., 2007 ; Moore et al., 2007 ) is 10 steps longer than the D & E molecular support for a relationship with eudicots needs to be tree (786 steps). When Archaefructus (inf) is added to the J/M tested by future combined analyses. However, it should be backbone tree, its most parsimonious position is sister to Cer- noted that bootstrap support for the link between Ceratophyl- atophyllum (791 steps; Fig. 2B ). Next best are positions linked lum and eudicots is only modest (71% in Moore et al., 2007 ; with Hydatellaceae (one step worse) and sister to core Nympha- 74– 89% in Saarela et al., 2007); that analyses by Moore et al. eales (two steps worse). (2007) using various methods and subsets of data gave different If pollen characters of Archaefructus are scored as unknown topologies, some with Chloranthaceae in a more basal position; (Archaefructus NP), its most parsimonious position with the and that other molecular analyses have linked Ceratophyllum D& E backbone (781 steps; not shown) is sister to the eudicot with Chloranthaceae (Duvall et al., 2006; Mathews, 2006; Qiu genus Euptelea (Ranunculales). Its next most parsimonious posi- et al., 2006). The strength of the morphological synapomor- tions (782 steps) are sister to Hydatellaceae, Ceratophyllum , Cer- phies might be questioned on the grounds that they largely rep- atophyllum plus Chloranthaceae, Ranunculales other than resent reductions and simplifi cations from ancestral states in Euptelea , Circaeaster (also Ranunculales), and the clade consist- angiosperms, which might be expected to give similar results ing of eudicots, monocots, and magnoliids. With the J/M back- regardless of their starting point. However, this is not in itself bone, omitting pollen characters strengthens the association of evidence that they are systematically worthless: without Cer- Archaefructus with Ceratophyllum (789 steps), which becomes atophyllum all these features are valid synapomorphies of Chlo- three steps rather than one step more parsimonious than its next- ranthaceae, which are independently supported as a clade by best positions (792 steps), which are sister to Hydatellaceae, molecular data. Euptelea , Ranunculales other than Euptelea , and Circaeaster . These results suggest the intriguing possibility that Cerato- When Archaefructus is scored as having a bisexual fl ower phyllum is an aquatic derivative of a terrestrial stem relative of ( Archaefructus fl o) and added to the D & E backbone tree, it has Chloranthaceae that already had many features of the crown three most parsimonious positions (783 steps; not shown): sis- group. Many additional changes would have to occur on the ter to Hydatellaceae, Cabomba , and core Nymphaeales. Seven line leading to Ceratophyllum: origin of a protoxylem lacuna positions are one step worse, including not only elsewhere in (2), loss of cambium (4), loss of pericyclic fi bers (6), dissection Nymphaeales but also sister to Magnoliales plus Laurales, of the leaves (20) and shift to dichotomous venation (18), loss Magnoliaceae, Circaeaster plus Lardizabalaceae, and Cir- of pollen aperture (62) (and almost total reduction of the exine: caeaster. When it is added to the J/M backbone tree, it has Takahashi, 1995), loss of stigmatic papillae (82), reduction or four most parsimonious positions (793 steps), including those found with the D & E backbone and as the sister group of fusion of the integuments to one (94), and large embryo (109). Ceratophyllum . Chloranthaceae and their extinct relatives are emerging as one Characters supporting these relationships of Ceratophyllum of the fi rst successful angiosperm lines ( Eklund et al., 2004 ; and Archaefructus are presented in the Discussion section. A Feild et al., 2004 ), which included greater diversity than would list of inferred ancestral states in angiosperms for fl oral charac- be inferred from the four living genera alone. Our results con- ters is presented in Table 1 , with differences among eight trees, cerning Ceratophyllum therefore raise the possibility that some involving all combinations of the D& E vs. J/M backbone trees, Early Cretaceous carpels or pollen that resemble Chloran- Amborella sister to all other angiosperms vs. Amborella and thaceae might actually be closer to Ceratophyllum and might Nymphaeales forming a clade, and exclusion vs. inclusion of therefore provide evidence on steps in its origin. Archaefructus. Parsimony optimizations of selected characters Our analysis provides provisional support for the speculative on various trees are presented in Figs. 3 – 12 . suggestion of Saarela et al. (2007) that Archaefructus is related to Hydatellaceae. This is the most parsimonious position of Archae- D ISCUSSION fructus when its fertile axis is interpreted as a raceme of unisexual fl owers and Ceratophyllum is associated with Chloranthaceae, as Phylogenetic results— Our inference that Ceratophyllum is with the D & E backbone. Unequivocal synapomorphies of the related to Chloranthaceae is supported by fi ve unequivocal sy- two groups are loss of fl oral subtending bracts (25) and loss of napomorphies: sessile fl ower (character 24), one stamen (40), perianth (31). The fact that the fl owers are unisexual is consistent embedded pollen sacs (51), one carpel (74), and orthotropous but not indicative because the polarity of this character is equivo- ovule (93). Synapomorphies of Chloranthaceae that are not cal. Other features of Archaefructus that support a relationship to found in Ceratophyllum and thereby place Ceratophyllum out- Nymphaeales as a whole are palmate venation (17) (reduced to side the family are sheathing leaf bases (12), interpetiolar one vein in Hydatellaceae), boat-shaped pollen (61), and palisade stipules (13), and stigmatic protuberances (81). Remarkably, it exotesta (101). This result would suggest that Hydatellaceae may is only one step less parsimonious to nest Ceratophyllum within be what became of one member of the Archaefructus group after Chloranthaceae, where its best position is sister to Hedyosmum , 125 Myr of further reduction in an aquatic habitat. January 2009] Endress and Doyle — Ancestral flowers 29

Table 1. Most parsimonious ancestral states for all characters concerning infl orescence and fl oral structure (see Appendix 1 for complete defi nitions), given different backbone trees (D& E vs. J/M), rooting with Amborella alone sister to all other angiosperms (A, N) vs. Amborella and Nymphaeales forming basal clade (A+N), and Recent taxa only vs. Recent taxa and fossil Archaefructus (R, F). When the reconstructed ancestral state is identical for all trees, it is given only once.

D&E J/M A,N R A+N R A,N F A+N F A,N R A+N R A,N F A+N F 22. Infl orescence 0 solitary, 1 botryoid etc., 2 raceme etc. 1/2 2 1/2 2 1/2 2 1/2 2 23. Infl orescence units 0 single fl ower, 1 cymes 0 solitary fl ower 24. Pedicel 0 present, 1 absent (sessile) 0 present 25. Bracts 0 present, 1 absent in male, 2 absent in all 0 present 26. Sex of fl owers 0 bisexual, 1 unisexual 0/1 bi/unisexual 27. Hypanthium 0 absent, 1 present, 2 inferior ovary 0/1 0 0/1 0 0/1 0 0/1 0 28. Receptacle 0 short, 1 elongate 0 short 29. Cortical vasculature 0 none or P, 1 A, 2 A plus G 0 none 30. Floral apex 0 used up, 1 protruding 0 used up 31. Perianth 0 present, 1 absent 0 present 32. Perianth phyllotaxis 0 spiral, 1 whorled 0/1 spiral/whorled 33. Perianth merism 0 trimerous, 1 dimerous, 2 polymerous 0 trimerous 34. Perianth whorls 0 one, 1 two, 2 more than two 2 more than two 35. Tepal differentiation 0 sepaloid, 1 sep + pet, 2 petaloid 0/1 1 0/1 1 0/1 1 0/1 1 36. Petals 0 absent, 1 present 0 absent 37. Inner perianth nectaries 0 absent, 1 present 0 absent 38. Outer perianth fusion 0 free, 1 fused 0/1 0 0/1 0 0/1 0 0/1 0 39. Calyptra 0 absent, 1 present 0 absent 40. Stamen number 0 more than one, 1 one 0 more than one 41. Stamen phyllotaxis 0 spiral, 1 whorled 0/1 spiral/whorled 42. Stamen merism 0 trimerous, 1 dimerous, 2 polymerous 0 trimerous 43. Stamen whorls 0 one, 1 two, 2 more than two 2 more than two 44. Stamen position 0 single, 1 double 0 single 45. Stamen fusion 0 free, 1 connate 0 free 46. Inner staminodes 0 absent, 1 present 0 absent 47. Food bodies 0 absent, 1 on staminodes 0 absent 48. Stamen base 0 short, 1 long wide, 2 long narrow 1/2 1/2 0/1/2 0/1/2 1/2 1/2 1/2 1/2 49. Paired basal stamen glands 0 absent, 1 present 0 absent 50. Connective apex 0 extended, 1 truncated, 2 peltate 0 0 0 0 0/1 0/1 0 0 51. Pollen sacs 0 protruding, 1 embedded 0 protruding 52. Microsporangia 0 four, 1 two 0 four 53. Orientation 0 introrse, 1 latrorse, 2 extrorse 0/1 0/1 0/1 0/1 0 0 0 0 54. Dehiscence 0 longitudinal slit, 1 H-valvate, 2 fl aps 0 longitudinal slit 74. Carpel number 0 more than one, 1 one 0 more than one 75. Carpel form 0 ascidiate, 1 intermediate, 2 plicate 0 ascidiate 76. Postgenital fusion 0 none, 1 partial, 2 complete 0 none 77. Secretion 0 present, 1 absent 0 present 78. PTTT a 0 not differentiated, 1 differentiated, 2 multilayered 0 not differentiated 79. Style 0 absent, 1 present 0 0/1 0/1 0/1 0 0/1 0 0/1 80. Stigma 0 extended, 1 restricted 0 extended 81. Stigmatic protuberances 0 absent, 1 present 0/1 0 0/1 0 0/1 0 0/1 0 82. Stigmatic papillae 0 none, 1 unicellular, 2 pluricellular 1/2 uni/pluricellular 83. Extragynoecial compitum 0 absent, 1 present 0 present 84. Fusion 0 apocarpous, 1 paracarpous, 2 eusyncarpous 0 apocarpous 85. Oil cells 0 not visible, 1 intrusive 0 not visible 86. Unicellular hairs on carpels 0 absent, 1 present 0 absent 87. Curved hairs on carpels 0 absent, 1 present 1 0/1 1 0/1 0 0/1 0 0/1 88. Abaxial nectaries 0 absent, 1 present 0 absent 89. Septal nectaries 0 absent, 1 present 0 absent 90. Ovule number 0 one, 1 mostly two, 2 more than two 0 0 0/2 0/2 0 0 0 0 91. Placentation 0 ventral, 1 laminar-dorsal 0 ventral 92. Ovule direction 0 pendent, 1 horizontal, 2 ascendent 0 pendent 93. Ovule curvature 0 anatropous, 1 orthotropous 0/1 0 0/1 0 0/1 0 0/1 0

apollen tube transmitting tissue. 30 American Journal of Botany [Vol. 96

In contrast, with the J/M backbone tree, in which Cerato- Archaefructus has racemes and Hydatellaceae have modifi ed phyllum is divorced from Chloranthaceae and associated with thyrses, as Rudall et al. (2007) argued, the order of fl owers in the eudicots, it is more parsimonious to associate Archaefructus two groups cannot be so easily compared. If the main axis of the with Ceratophyllum , based on dissected leaves (20), dichoto- infl orescence in Hydatellaceae (as reconstructed by Rudall et al., mous venation (18), loss of fl oral bracts (25), unisexual fl owers 2007, in fi g. 5D) is compared with the main axis in Archaefruc- (26), and loss of perianth (31). This position is four steps less tus , there is no difference in the relative position of male and fe- parsimonious with the D & E backbone, where Ceratophyllum is male fl owers in the two groups. In both, the male fl owers (plus associated with Chloranthaceae, which have more features that female fl owers in Hydatellaceae) are borne on more basal lateral confl ict with those of Archaefructus , such as opposite leaves units (cymes in Hydatellaceae), while the more distal lateral units (9), pinnate venation (17), round pollen (61), and reticulate tec- are entirely female. Furthermore, the argument that an opposite tum (66). Better evidence on the position of Ceratophyllum order of male and female fl owers precludes a relationship is not could therefore have an impact on the best interpretation of Ar- compelling because analogies with other groups suggest that the chaefructus. Our results also depend on uncertain assumptions order of fl owers in bisexual infl orescences can reverse. For ex- concerning the morphology of the fertile structures of Archae- ample, in Buxaceae, male fl owers are basal and female fl owers fructus. When the fertile shoot is interpreted as a fl ower or pre- terminal in Buxus and Styloceras kunthianum, female basal and fl ower (Sun et al., 2002), which we regard as unlikely, one of male apical in Sarcococca and Pachysandra , and infl orescences the most parsimonious positions of Archaefructus is still with are unisexual in other Styloceras species. Based on inferred phy- Hydatellaceae, but it is equally parsimonious to place it else- logenetic relationships ( von Balthazar and Endress, 2002b ), ei- where in Nymphaeales. Confi rmation of the view of Ji et al. ther one or the other bisexual condition could be ancestral, but (2004) that the seeds of Archaefructus were orthotropous would the other bisexual type would be derived from it. increase the relative parsimony of a link with Ceratophyllum . Some of the evidence for a relationship of Archaefructus Ancestral fl oral states and initial specializations— In the with Hydatellaceae comes from the report by Sun et al. (2001, following sections, we consider the ancestral state reconstruc- 2002 ) of boat-shaped, tectate monosulcate pollen grains in Ar- tions in Table 1 and their general implications. Contrary to chaefructus, which was questioned by Friis et al. (2003). With some expectations (e.g., Qiu et al., 2006), trees in which Ambo- the D& E backbone, removal of pollen characters weakens the rella is sister to all other angiosperms and those in which it is connection of Archaefructus with Hydatellaceae and favors a linked with Nymphaeales have only modestly different impli- link with the eudicot genus Euptelea, supported in part by ab- cations for ancestral states: all seven differences involve cases sence of a perianth (31) and one stamen whorl (43), as well as in which the ancestral state is equivocal with one rooting and palmate venation (17), shared with eudicots as a whole, and one of the same two states with the other. Addition of Archae- several ovules (90), a synapomorphy of mesangiosperms other fructus has even less impact, with a few important exceptions to than Chloranthaceae and Ceratophyllum . The possibility that be discussed. Finally, except for the positions of Chloranthaceae Archaefructus was related to eudicots was raised by Friis et al. and Ceratophyllum, the differences between arrangements of (2003), based especially on the ternate, dissected leaf architec- mesangiosperm lines in the D & E combined and J/M plastid ture. Such a relationship would imply that Archaefructus had trees ( Jansen et al., 2007 ; Moore et al., 2007 ) have generally tricolpate rather than monosulcate pollen, which would be sur- minor effects. This result seems due to two factors. First, infer- prising in light of its Barremian-Aptian age, when tricolpate ences on ancestral states are most dependent on relationships in pollen was exceedingly rare outside northern Gondwana (Doyle, the ANITA grade, which are the same with both arrangements. 1992; Hughes, 1994; Hochuli et al., 2006). However, even in Second, very few morphological changes occurred on the inter- the absence of pollen characters, relationships with Hydatel- nodes between the three main lineages of mesangiosperms laceae and Ceratophyllum remain almost as parsimonious with (magnoliids, eudicots, and monocots), however they are ar- the D& E backbone, and the link with Ceratophyllum is strength- ranged, presumably because these lineages radiated in a very ened with the J/M backbone. These results underline the need short time (Moore et al., 2007), the same reason their relation- for more convincing evidence on pollen of Archaefructus . ships have been so diffi cult to resolve. Our analysis does not address the hypothesis that Archae- fructus is a stem relative of all living angiosperms rather than a Infl orescence organization— Because of varying views on member of the crown group (Sun et al., 2002): it only specifi es interpretation of fl owers and infl orescences in taxa such as Ar- the most parsimonious position(s) of Archaefructus if it belongs chaefructus, Hydatellaceae, and Chloranthaceae and recent in the crown group. However, the crown group hypothesis was suggestions that the distinction between infl orescences and supported by an analysis of living and fossil seed plants (Doyle, fl owers may be labile or problematic in basal angiosperms ( Friis 2008), including all the ANITA lines, Chloranthaceae, and and Crane, 2007; Rudall et al., 2007), we have considered char- three magnoliids. When Archaefructus was interpreted as hav- acters of infl orescences as well as fl owers. ing an infl orescence of unisexual fl owers, its most parsimoni- Based on our results, with Amborella basal ( Fig. 3 ), the an- ous position was with Hydatellaceae, and a position sister to all cestral infl orescence type (character 22 ) in angiosperms is living angiosperms was fi ve steps worse. When the fertile axis equivocal: either botryoids, as in Amborella; or racemes (which was interpreted as a bisexual fl ower, it was again more parsimo- some authors might describe as stems with solitary axillary nious to place Archaefructus in Nymphaeales than below living fl owers), as in Nymphaeales, Chloranthaceae (modifi ed to angiosperms, but by three steps rather than fi ve. spikes and thyrses), and basal eudicots and monocots. However, Rudall et al. (2007) cited the order of fertile parts in Archae- if Amborella is linked with Nymphaeales, the ancestral type can fructus (stamens basal, carpels apical) as an argument against a be reconstructed as a raceme. Both hypotheses imply that soli- relationship with Hydatellaceae, where the female fl owers in spe- tary fl owers, often considered ancestral in angiosperms, are in- cies with bisexual infl orescences are to the outside (assumed stead derived: from racemes in Austrobaileyales (with a shift to to be basal) and male fl owers are central (apical). However, if botryoids in Trimenia ), magnoliids, and Nelumbo , and from January 2009] Endress and Doyle — Ancestral flowers 31 botryoids in the Hydrastis -Glaucidium clade in Ranunculaceae. the base of the pedicel, but she argued that the Nuphar condition In magnoliids, solitary fl owers may have evolved either once is derived, as a result of intercalary growth between the abaxial from racemes at the base of the Magnoliales-Laurales clade, tepal and the rest of the fl ower. This interpretation is less plausi- with a reversal in Myristicaceae and a shift to botryoids in Lau- ble in terms of outgroup comparison. We have therefore scored rales, or separately from racemes in Magnoliales and from ei- both Nuphar and Nymphaeoideae (Nymphaea , Euryale , and Vic- ther racemes or botryoids in Laurales (Calycanthaceae). toria) as having racemes, with the fl oral subtending bract present Thyrses, distinguished from racemes and spikes by the in Nuphar but absent in Nymphaeoideae (which could be due lateral unit character ( 23; cymes rather than single fl owers), either to reduction or to incorporation into the perianth). The appear to be derived from racemes in Hydatellaceae, Chloran- condition in Barclaya is unknown, although it appears consistent thaceae, Aristolochioideae, and Butomus , and from either ra- with that in Nuphar and Nymphaea . cemes or botryoids in Siparunaceae and Hernandiaceae. Sessile From this perspective, the whole shoot system of Nymphae- fl owers ( 24) were derived from pedicellate ones, resulting in aceae can be considered a giant raceme. If the pherophyll-bud spikes in Chloranthaceae and Ceratophyllum (a synapomorphy primordium is viewed as a complex of two parts (cf. Chassat, with the D & E backbone, a convergence with the J/M back- 1962 ), in some cases, the pherophyll part develops into a foliage bone), the Piperaceae-Saururaceae clade, monocots (separately leaf, and the fl oral bud is suppressed; in others, the fl oral bud in Acorus , Araceae, and Aponogeton ), and Platanus (modifi ed grows rapidly after initiation, and the pherophyll is reduced to a into heads), and botryoids with sessile fl owers (i.e., stachyoids) thin bract or nothing at all. One could also suggest there is com- in Tetracentron . In all these cases, reduction of the pedicel is petition for space: either the fl ower or the leaf is reduced, and the correlated with general fl oral reduction. Loss of bracts ( 25 ; ar- other, more precocious part “ wins. ” This divergence in develop- rows in Fig. 3) occurred in several lines in which racemes were ment may be a function of the gigantism of the shoots, leaves, modifi ed to spikes (Hedyosmum and Ceratophyllum , either and fl owers of these plants, compared to their outgroups. once or twice, depending on backbone tree and optimization; Victoria and Euryale may provide indirect support for this Acorus , Araceae, Aponogeton , Platanus ) or thyrses of reduced interpretation. Borsch et al. (2007) identifi ed these taxa as the pedicellate fl owers (Hydatellaceae) and might also seem corre- sister group of Nymphaea , but subsequent analyses of more lated with reduced fl owers. However, this is not a universal rule markers (L ö hne et al., 2007) indicate they are nested within because bracts were also lost within Nymphaeaceae, in which Nymphaea and are therefore unlikely to represent the ancestral fl owers are unusually large. condition in Nymphaeaceae. However, they too can be inter- Nymphaeales deserve special attention because interpreta- preted in terms of an underlying racemose pattern. In Victoria tion of their infl orescence morphology is both particularly con- and Euryale the fl owers arise in a Fibonacci spiral. Each fl ower troversial and potentially relevant to ancestral conditions and is associated with a leaf, but it is located not in the middle of the early trends in angiosperms. Cutter (1957a, b , 1959 , 1961 ) de- leaf axil but rather toward the inner side, in terms of the direc- scribed Nymphaeaceae (Nuphar , Nymphaea) as having a unique tion of the spiral (anodic side; Cutter, 1961 ; Schneider et al., system of solitary fl owers borne in the same phyllotactic spiral 2003). Cutter (1961) described the leaves and fl owers as form- as leaves, with no subtending bracts (accepted by Schneider et ing two separate spirals, but an interpretation more consistent al., 2003 ), which she compared with conditions in ferns. This with normal angiosperm morphology may be that each fl ower is view was critiqued by Chassat (1962), who interpreted Nympha- in the axil of a foliage leaf but slightly displaced ( Chassat, eaceae as having modifi ed racemes, with the apparent position 1962 ). This displacement might be due to the fact that both leaf of fl owers in the same spiral as leaves due to reduction of the (petiole) and fl ower (pedicel) are bulky, so an exact superposi- leaf (pherophyll) component of a leaf-bud primordium. tion would not allow enough space in the mature state. As in In a phylogenetic context, with Cabombaceae sister to Nymphaea , the abaxial tepal in Victoria develops fi rst; but the Nymphaeaceae, the interpretation of Chassat (1962) makes more fact that Victoria has a subtending leaf as well could be evi- sense because Cabomba has racemes, with fl owers borne in the dence against identifi cation of the abaxial tepal in Nymphaea axils of peltate fl oating leaves ( Brasenia has not been studied in with the fl oral subtending bract. Because each pherophyll de- suffi cient detail for comparisons). It would also bring Nympha- velops into a leaf and each bud develops into a fl ower, the num- eaceae in line with the normal shoot organization in angiosperms ber of fl owers and leaves in a shoot is the same. In contrast, in and other seed plants. Closer examination of infl orescence and Nuphar and Nymphaea only the leaf or only the fl ower of the fl oral morphology in Nymphaeaceae supports this view. Nuphar , pherophyll/fl ower “ complex ” develops to maturity, and the which is basal in Nymphaeaceae, has a bract near the base of the numbers of mature fl owers and leaves in a shoot are not neces- pedicel, on its abaxial side with respect to the main axis and thus sarily equal. If Victoria and Euryale are nested in Nymphaea , in near the position of a subtending bract, and three outer tepals. which the subtending leaf is absent, their condition may repre- Nymphaea , however, has no bract on the pedicel and four outer sent a “ reactivation ” of the pherophyll portion of the leaf-bud tepals, with the fi rst-formed tepal abaxial relative to the main primordium, perhaps related to even more extreme gigantism. axis, like the bract in Nuphar. As discussed by Chassat (1962), In Hydatellaceae, interpretation of the crowded infl ores- this structure might be derived from that in Nuphar either by cences of extremely simple fl owers is made diffi cult by the lack complete reduction of the subtending bract or by its incorpora- of subtending bracts for the lateral branches. However, Rudall tion into the perianth as the abaxial tepal. The latter hypothesis et al. (2007) tentatively but plausibly interpreted the fl owers as would explain the change from trimerous to tetramerous organi- forming reduced thyrses. zation of the perianth. On the other hand, the earlier development Based on the inferred phylogenetic relationships, racemes of the abaxial tepal could be a function of the fact that the fl ower are ancestral in Nymphaeales, either as a synapomorphy or a is more developed on the abaxial side at the time the tepals are retention from the fi rst angiosperms. With bracts present in initiated and somewhat incurved. Cutter (1957b) also homolo- Cabomba and Nuphar, it is most parsimonious to assume that gized the bract in Nuphar with the abaxial tepal in Nymphaea , bracts were lost independently on the line to Hydatellaceae and noting cases in Nymphaea in which this tepal is displaced toward Archaefructus (if these two taxa form a clade) and within 32 American Journal of Botany [Vol. 96

Fig. 3. D & E tree of Recent taxa, with coloring of branches showing most parsimonious course of evolution of infl orescence character (22; state 1 = botryoid, panicle, or thryrsoid; state 2 = raceme, spike, or thyrse). Boxes under names of taxa indicate their character state; colors of branches indicate their reconstructed state based on parsimony optimization. Arrows indicate losses of fl oral subtending bracts (character 25) and possible states on branches where parsimony optimization is equivocal (e.g., 0/2 = either solitary fl ower or raceme). Position and number of losses of bracts in Ceratophyllum and Chloranthaceae are equivocal. Abbreviations as in Fig. 2 .

Nymphaeaceae (Nymphaea ). Archaefructus still had racemes, leading to the remaining mesangiosperms is bisexual. With the but these were modifi ed into thyrses in Hydatellaceae, by re- J/M backbone and Amborella basal, the state is equivocal up to placement of single lateral fl owers by cymes. One possible the node connecting Nymphaeales and the remaining groups. adaptive explanation is that the resulting increase in number of Rescoring Archaefructus as uncertain (0/1) for this character fl owers compensated for the reduction in number of carpels and based on the report of bisexual units by Ji et al. (2004) would ovules, but the small number of cymes in the living group may not modify these inferences. refl ect a later round of reduction. The view that bisexual fl owers were ancestral is supported by the regular presence of one or two sterile stamens in female Floral organization— In recent years most authors have as- fl owers of Amborella ( Endress and Igersheim, 2000b ; Buzgo et sumed that the fi rst angiosperms had bisexual fl owers ( 26 ), but al., 2004). In other words, the fl owers are organizationally bi- because the fl owers of Amborella are functionally unisexual the sexual. Michael Frohlich, Royal Botanic Gardens, Kew (per- ancestral state is equivocal. With our previous data set, the lin- sonal communication) also gave us a likelihood argument in eage leading to all other angiosperms could be reconstructed as support of the view that the unisexual state in Amborella is de- basically bisexual, but the situation has changed with the addi- rived, namely that Amborella terminates a long branch with no tion of Hydatellaceae (and Archaefructus , if it is linked with surviving side-branches, whereas the sister branch (including Hydatellaceae). With the D & E backbone, the state is equivocal all the remaining angiosperms) is “ broken up” by several lin- up to the basal node of the mesangiosperms, above which the eages near its base. This difference in branch length could mean Chloranthaceae-Ceratophyllum line is unisexual and the line that inference of the initial state is more secure on the latter line January 2009] Endress and Doyle — Ancestral flowers 33

Fig. 4. (A) D& E tree, showing inferred evolution of perianth phyllotaxis (character 32). Arrows indicate loss of perianth (character 31); position and number of losses in Ceratophyllum and Chloranthaceae are equivocal. (B) Inferred evolution of perianth merism (character 33); taxa with spiral phyllotaxis scored as unknown. Abbreviations as in Fig. 2 . 34 American Journal of Botany [Vol. 96 than on the longer line leading to Amborella . However, this ar- perianth). Independent losses occurred in Hydatellaceae (with gument is weakened by the addition of Hydatellaceae (either or without Archaefructus ), the Piperaceae-Saururaceae clade, with or without Archaefructus ) to Nymphaeales. the strange case of Eupomatia (and Galbulimima if its outer An intriguing case concerns Chloranthaceae, in which petaloid organs are staminodes; we scored perianth as unknown, Hedyosmum and Ascarina have unisexual fl owers, but Sarcan- but it is probably absent), and Euptelea (arrows in Fig. 4A ). dra and Chloranthus have bizarre bisexual fl owers consisting Loss of the perianth in Ceratophyllum and Chloranthaceae of one carpel and one stamen or tripartite androecium ( Endress, other than Hedyosmum poses more problems. The analyses of 1987b; Eklund et al., 2004). Molecular studies and the morpho- Doyle and Endress (2000) and Eklund et al. (2004) indicated logical analysis of Eklund et al. (2004) agree that Hedyosmum that the presence of three tepals in Hedyosmum was plesiomor- and Ascarina diverged successively below Sarcandra and phic and their loss a synapomorphy of other Chloranthaceae, Chloranthus . In Eklund et al. (2004) , in which Chloranthaceae and this is still so for the J/M tree. However, if Ceratophyllum were nested among bisexual taxa, there were two equally parsi- is related to Chloranthaceae, as in the D& E tree (Fig. 4A), and monious scenarios: either bisexual fl owers were plesiomorphic if we are correct in interpreting Ceratophyllum as lacking a pe- for the family and became unisexual independently in Hedyos- rianth, it is equivocal whether the perianth of Hedyosmum is a mum and Ascarina , or fl owers became unisexual in the ancestor primitive retention or a secondary invention. of the family and reverted to bisexual in Sarcandra and Chlo- As discussed in Endress and Doyle (2007), the ancestral pe- ranthus (Doyle et al., 2003). This is still true in trees with the rianth phyllotaxis ( 32 ; Fig. 4A ) is equivocal: either the spiral J/M backbone ( Fig. 2B ). However, in trees with the D & E back- state of Amborella and Austrobaileyales is ancestral and the bone (Fig. 2A), where Chloranthaceae are linked with Cerato- whorled state of Nymphaeales is derived, or vice versa. How- phyllum, which has unisexual fl owers, it is most parsimonious ever, the reconstructed ancestral state in mesangiosperms is un- to assume that the bisexual fl owers of Sarcandra and Chloran- ambiguously whorled (see also Zanis et al., 2003). Cases of thus were derived from unisexual fl owers. In “ higher ” angio- spiral perianth in magnoliids, once widely assumed to be primi- sperm groups, we know of no cases where phylogenetic analyses tive, are therefore derived from whorled: in Degeneria in the imply that bisexual fl owers are derived, but it would be danger- Magnoliales, and once, twice, or three times in Laurales. In the ous to assume this was true during the early angiosperm radia- eudicots, shifts to spiral occurred in Circaeaster , core Ranun- tion. In any case, it appears that fl oral sexuality was highly culaceae, and Nelumbo . If a spiral perianth originated once at labile in early angiosperms. At least eight reversals from bi- the base of Laurales and was retained into Calycanthaceae, sexual to unisexual also occurred within magnoliids and basal Atherospermataceae, Gomortega , and the monimiaceous genus eudicots: in Lactoris (some fl owers), Myristicaceae, Sipar- Hortonia , there was yet another round of reversal, from spiral unaceae, Mollinedioideae and Monimioideae (once or twice), to whorled, in other Monimiaceae and the Lauraceae-Hernandi- Lardizabalaceae, Menispermaceae, Platanus , and Buxaceae. aceae clade. As argued by Endress (1987a), perianth phyllotaxis Congenital fusion of all outer fl oral parts into a hypanthium is therefore a highly labile character, although it is stable over ( 27) occurred independently in Amborella (where it could be large parts of the tree. either ancestral or derived if Amborella alone is basal, but de- Tracing the evolution of perianth merism ( 33 ; Fig. 4B ) is rived if Amborella and Nymphaeales form a clade), Eupomatia , potentially confused by the occurrence of many taxa with spiral and Laurales, where it is an important synapomorphy of the phyllotaxis, in which merism was scored as unknown but parsi- order. We treated inferior ovary as a state of the same character, mony optimization implicitly treats taxa as having the state of on the grounds that it might originate by fusion of either sepa- the surrounding groups (cf. Maddison, 1993). Based on those rate outer parts or an existing hypanthium to the ovary. The taxa that are whorled, if the ancestral angiosperms had a whorled latter process does appear to have occurred in Laurales, where perianth, it was trimerous. It became polymerous within Gomortega and the clade consisting of Lauraceae (where the Nymphaeaceae (specifi cally tetramerous) and in Hernandiaceae ancestral state appears to be inferior, as in Hypodaphnis and (Gyrocarpoideae, some Hernandioideae), dimerous in Winter- other basal genera: Rohwer and Rudolph, 2005) and Hernandi- aceae. Whether the shift from trimerous to tetramerous perianth aceae are nested within the order. However, there is no phylo- in Nymphaeaceae was a result of incorporation of the bract into genetic evidence for this in the other lines with an inferior ovary the perianth, as discussed, would be an intriguing topic for evo- ( Barclaya plus Nymphaeoideae, Hedyosmum , Saururaceae, devo studies. Most interesting is the case of eudicots, in which Aristolochiaceae, Dioscoreaceae, and Trochodendraceae), the reconstructed ancestral state is either trimerous, as in most whose closest outgroups have no hypanthium. Ranunculales, or dimerous (a possibility fi rst emphasized by An elongate receptacle (28 ), often presented as a primitive Drinnan et al., 1994), as in Papaveraceae, near the base of Ra- feature, appears instead to be an independent advance of nunculales, and in Proteaceae, Tetracentron (Chen et al., 2007), Schisandraceae, Magnoliaceae, and Galbulimima . In Magnoli- and Buxaceae (von Balthazar and Endress, 2002a), on the line aceae this coincided with origin of cortical vasculature ( 29 ) leading to “ core ” eudicots ( Gunneridae , including Pentapeta- extending from the perianth into the gynoecium, a feature that lae , of Cantino et al., 2007 ). arose independently in Glaucidium . Cortical vasculature ex- The fact that trimery is reconstructed as homologous in tending only into the androecium arose before an elongate re- Hedyosmum (with three tepals) and other groups (Fig. 4B) ceptacle in the Degeneria - Galbulimima clade and independently might be questioned as an artifact of the Maddison long dis- in Trochodendron . Protrusion of the fl oral apex ( 30 ), a dis- tance effect ( Maddison, 1993 ). With the J/M backbone, where tinctive feature of Nymphaeoideae and Illicium , is an indepen- the presence of a perianth in Hedyosmum is reconstructed as dent advance in these two groups. ancestral, this poses no problem. However, with the D& E back- bone ( Fig. 4B ), where Chloranthaceae are linked with Cerato- Perianth— Our results indicate that presence of a perianth phyllum, the perianth of Hedyosmum may be a secondary ( 31) is ancestral, even with the addition of Archaefructus , which invention, and if so its trimery would not be strictly homolo- has no perianth (both of its potential extant relatives also lack a gous with trimery in other taxa. On the other hand, it might be January 2009] Endress and Doyle — Ancestral flowers 35

Fig. 5. (A) D & E tree, showing inferred evolution of number of perianth whorls (series in taxa with spiral phyllotaxis; character 34). (B) Inferred evolu- tion of tepal differentiation (character 35; state 1 = outer sepaloid, inner petaloid). Abbreviations as in Fig. 2 . 36 American Journal of Botany [Vol. 96 that the reappearance of a perianth in trimerous form was a con- At least basal fusion of the outermost perianth parts ( 38 ) oc- sequence of reactivation of an existing but suppressed develop- curred independently in several lines: Amborella (there is a mental program and therefore homologous at a more fundamental short zone of fusion among tepals before fusion with the sta- genetic level (cf. Li et al., 2005 ). This reappearance of trimery mens begins), Cabomba (Endress, 2008a), Canellales and Aris- could be an intriguing topic for developmental genetic research tolochiaceae (either once or twice), Myristicaceae, and (cf. “ biological homology” of Wagner, 1989, 2007 ). But even if Degeneria (Magnoliales). On parsimony grounds, the tepal fu- the inferred homology of the trimerous perianth in Hedyosmum sion in Amborella could be ancestral if Amborella is sister to all is an artifact, it would still be valid to conclude that trimery is other angiosperms, but it is derived if Amborella and Nympha- ancestral in angiosperms (if they were originally whorled) be- eales form a clade. This fusion is not related to formation of a cause this is also inferred if Hedyosmum is deleted. calyptra (39 ), apparently derived from one or two fl oral bracts The ancestral number of perianth whorls (series when spi- ( Endress, 1977 , 2003 ; Kim et al., 2005a ), which intriguingly ral) ( 34 ) is reconstructed as more than two, and this was re- arose either once or three times in other Magnoliales (Magnoli- tained from the fi rst angiosperms into magnoliids ( Fig. 5A ). aceae, Galbulimima , Eupomatia). At the level of angiosperms The number of whorls was reduced to two in Cabombaceae, as a whole, fusion tends to be much more labile in sepals (or Lauraceae, and (independently or as a synapomorphy with outer tepals) than in petals. This phenomenon is refl ected by Lauraceae) some Hernandiaceae. With the D& E backbone the fact that the contrast of choripetaly vs. sympetaly has been (Fig. 5A), a shift to two whorls is a conspicuous synapomor- commonly regarded as signifi cant at the macrosystematic level, phy of monocots and occurred two or three times in eudicots — whereas chorisepaly vs. synsepaly has been relatively neglected. once in Ranunculaceae, and once or twice in the other branch, Among basal angiosperms, tepal fusion may be interesting depending on whether the numerous series in Nelumbo are an- within genera or families, for example in Hedyosmum , where it cestral or derived. However, with the J/M backbone, where unites a large derived clade ( Eklund et al., 2004 ). Another inter- monocots, Ceratophyllum, and eudicots form a clade, reduc- esting feature concerns the fate of tepals after anthesis in the tion to two whorls may be either an event that occurred three ANITA grade: caducous during or at the end of anthesis (com- or four times, or a synapomorphy of these groups, with two bined with narrow attachment areas) in Austrobaileyales, but reversals in eudicots. In Ranunculaceae, reduction to two persistent (combined with broad attachment areas) in Ambo- whorls appears to have been a step toward reduction to one in rella and Nymphaeales (Endress, 2008a). This feature has not Hydrastis, and the same may have occurred in Gyrocar- been explored throughout basal angiosperms and was therefore poideae. But one whorl was apparently derived directly from not used in our analysis. However, a caducous perianth may be more than two in Hedyosmum (if its perianth is ancestral in a synapomorphy of Austrobaileyales. The cases of tepal fusion Chloranthaceae), the Lactoris -Aristolochiaceae clade, Myris- appear to be restricted to clades with persistent tepals. ticaceae, and Circaeaster . The inferred ancestral state of perianth differentiation ( 35 ; Androecium— Our results indicate that the single stamen Fig. 5B) is either all sepaloid tepals, as in Amborella , or outer ( 40) of Hydatellaceae, Ceratophyllum , and most Chloran- sepaloid and inner petaloid tepals, the basic state in the line lead- thaceae is derived and is a synapomorphy of Ceratophyllum ing to all other angiosperms. If Amborella is linked with and Chloranthaceae with the D & E trees. Our data imply that the Nymphaeales, the differentiated state is unequivocally ancestral. presence of a few stamens in some species of Ascarina (scored Tepals became all sepaloid in Trimenia and one to three times in as 0/1) and the tripartite androecium of Chloranthus (scored as Laurales. As with the origin of two perianth whorls, with the unknown) were derived within Chloranthaceae. This inference D& E trees tepals became all sepaloid in monocots and once is sensitive to the interpretation of the androecium of Chloran- or twice in eudicots (Platanus , Proteaceae, Tetracentron , and thus , which has been variously considered a result of lobation Buxaceae), depending on whether the differentiated tepals of of one stamen or fusion of three ( Endress, 1987b ; Doyle et al., Nelumbo are ancestral or derived, but with the J/M trees this is a 2003). If Chloranthus is rescored as having more than one sta- possible synapomorphy of the two clades. Differentiated tepals men, and if Ceratophyllum is not related to Chloranthaceae (as became all petaloid in Cabombaceae and some Ranunculales. In in the J/M topology), the inferred ancestral state for the family monocots, the all petaloid state is a synapomorphy of the “ core ” is equivocal. However, if Ceratophyllum is associated with monocot clade represented by Dioscoreaceae, Nartheciaceae, Chloranthaceae, one stamen is reconstructed as ancestral. and Melanthiaceae (Petrosaviidae of Cantino et al., 2007), Stamen phyllotaxis ( 41 ; Fig. 6A ) is generally correlated apparently derived from all sepaloid. True petals ( 36) are a sy- with perianth phyllotaxis, but this correlation breaks down in napomorphy of Ranunculales other than Euptelea , with conver- Magnoliales ( Endress and Doyle, 2007 ). As with the perianth, gent origins in Nuphar and within Asaroideae (Saruma ). The the ancestral stamen phyllotaxis is equivocal, and with the D& E adaxial nectar glands ( 37 ) on the inner petals of many Ranun- topology the basic state for the magnoliid-monocot-eudicot culales apparently originated once after origin of petals, after clade (mesangiosperms other than Chloranthaceae and Cerato- divergence of Papaveraceae, with a convergence in Cabomba . phyllum , which cannot be scored) is whorled. However, Mag- If core eudicots ( Gunneridae of Cantino et al., 2007) are noliaceae have a whorled perianth (except for some probably linked with Buxaceae and/or Trochodendraceae ( Soltis et al., derived species of Magnolia) but spiral (or somewhat irregu- 2003 ), our results support the hypothesis that the typical dicy- larly arranged) stamens. Stamens are also spiral in Eupomatia clic, pentamerous perianth of the gunnerid groups other than and Galbulimima (which have no perianth), as are both tepals Gunnerales (Pentapetalae of Cantino et al., 2007) was derived and stamens in Degeneria . Myristicaceae have three whorled from two dimerous whorls of reduced sepal-like organs (con- tepals, but their fused stamens vary between spiral and whorled trary to Wanntorp and Ronse De Craene, 2005). Whether this (scored as 0/1). As a result, stamen phyllotaxis in magnoliids occurred by increase in the number of parts per whorl or by ad- appears to have shifted to spiral earlier than perianth phyl- dition and reorganization of new whorls is beyond the scope of lotaxis, in the common ancestor of Magnoliales and Laurales, this paper. with reversals to whorled in Annonaceae (often becoming January 2009] Endress and Doyle — Ancestral flowers 37

Fig. 6. (A) D& E tree, showing inferred evolution of androecium phyllotaxis (character 41). (B) Inferred evolution of number of stamen whorls (series in taxa with spiral phyllotaxis; character 43). Abbreviations as in Fig. 2 . 38 American Journal of Botany [Vol. 96 chaotic within the androecium: Endress, 1987a) and either once Eupomatia, and Annonaceae (where inner staminodes are re- or twice in Mollinedioideae and the Lauraceae-Hernandiaceae tained in the basal genus Anaxagorea; Maas and Westra, 1984; clade. With the J/M backbone, because whorled eudicots and Scharaschkin and Doyle, 2006). Glandular food bodies (47 ) on monocots are consolidated in a clade and Chloranthaceae are the inner staminodes are a more secure synapomorphy of the linked with magnoliids, the ancestral state in mesangiosperms Degeneria -Annonaceae clade. is equivocal, and it is possible that the spiral androecium in “ Laminar ” or “ leafl ike” stamens have often been considered Magnoliales and Laurales was retained from the fi rst angio- ancestral in angiosperms (e.g., Canright, 1952). However, the sperms, rather than being a reversal or a convergence with Am- fact that the sporangia are adaxial in some laminar stamens and borella and Austrobaileyales, as we inferred for the perianth. A abaxial in others suggests that the laminar condition may not be less consequential discrepancy between perianth and androe- homologous across basal angiosperms. This led Takhtajan cium occurs in Nelumbo, where tepals are spiral but stamens are (1969) to suggest that the ancestral stamen had marginal spo- produced chaotically on a ring primordium (Hayes et al., 2000; rangia, a condition usually associated with a narrow connec- here scored as unknown). tive. Rather than contrasting laminar and fi lamentous, we have As with the perianth, a trimerous androecium ( 42) is pre- split stamen morphology into several characters. dominant and reconstructed as ancestral (if one assumes the One character concerns the stamen base ( 48 ; Fig. 7A ), with androecium was originally whorled). Changes in stamen mer- three states: short (either wide or constricted, which often inter- ism are only sometimes correlated with those in the perianth. grade: Eklund et al., 2004), long and wide, and long and narrow The androecium became polymerous before the perianth in (= typical fi lament; see Appendix 1 for limits between states). Nymphaeaceae (before rather than after divergence of Nuphar ) Stamens with both of the fi rst two states have been described as and in Canellales, where the perianth remained trimerous in laminar. With our previous data set ( Doyle and Endress, 2000 ), Canellaceae (the polymerous androecium may be related to the ancestral state was either long and wide (as in Amborella connation of the stamens) and became dimerous in Winter- and most Austrobaileyales) or narrow (as in Cabombaceae and aceae. Like the perianth, the ancestral androecium in eudicots Trimenia ). This is still true with Recent taxa only and both may have been either trimerous or dimerous (the latter would backbone topologies, because Hydatellaceae also have a long be favored if Euptelea is dimerous, as some have suggested, but and narrow base, but with the D& E backbone and the addition this is unclear: Hoot et al., 1999 ; Ren et al., 2007 ). of Archaefructus , which has a short base, any of the three states The inferred ancestral number of stamen whorls (or series) may be ancestral. Within Nymphaeales, there is a series from ( 43 ; Fig. 6B ) is more than two, as in the perianth. However, in long and narrow to short to long and wide if only living taxa are contrast to the situation in the perianth, where more than two considered, but if Archaefructus is linked with Hydatellaceae whorls were apparently retained from the fi rst angiosperms into this becomes equivocal. With the D& E backbone, a long and magnoliids, with the D & E backbone a shift to two stamen narrow fi lament is basic for mesangiosperms other than the whorls occurred near the base of mesangiosperms (either above Chloranthaceae-Ceratophyllum line (most of which have a or below Chloranthaceae) and reversed to more than two in short base), and a shift to laminar stamens with a short base Magnoliales and Laurales (Fig. 5B). With the J/M backbone unites Magnoliales and Laurales. This reversed to long and nar- both this scenario and persistence of more than two stamen row in the clade consisting of Monimiaceae, Lauraceae, and whorls (or series) into magnoliids are equally parsimonious (as Hernandiaceae. However, with the J/M backbone, the state at was also true for spiral stamen phyllotaxis). With this backbone, the base of mesangiosperms is entirely unresolved, and the origin of two stamen whorls may be a synapomorphy of mono- short base of Magnoliales and basal Laurales may be either de- cots and eudicots, but it may equally well be homologous with rived or inherited from lower in the tree. the same state in Piperales and Canellaceae. Other noteworthy Paired basal glands ( 49 ), a peculiar feature of the stamens changes include increases from two to more than two stamen of many Laurales, originated either once after divergence of whorls (or series) in Ranunculaceae, Nelumbo , and Trochoden- Calycanthaceae, with reversals in Siparunaceae and Molline- dron, and reductions from more than two to one in Cabomba dioideae, or twice, in the Atherospermataceae-Gomortega and and Myristicaceae, from more than two to two in Hernandi- Monimiaceae-Lauraceae-Hernandiaceae clades. Given the dis- aceae, and from two to one in Euptelea and Circaeaster . tinctive nature of this advance and the fact that its absence in Production of stamens in double positions (44 ) evolved in- Siparunaceae and Mollinedioideae is correlated with packing of dependently in Nymphaeales (the condition in Hydatellaceae the stamens in a deep hypanthium, the former scenario may be and Archaefructus is undefi ned), Aristolochiaceae, Annonaceae, more likely. Butomus, and Papaveraceae, and within Winteraceae, Molline- An extended connective apex ( 50 ) is also common in lami- dioideae, and Tofi eldiaceae, where both states occur. At the nar stamens. As in Doyle and Endress (2000), this feature is taxonomic level of this analysis, stamen fusion (45 ) is a sepa- ancestral on most trees, except the J/M trees with Recent taxa rate advance wherever it occurs (Schisandraceae, Canellaceae, only, where the ancestral state is equivocal. If the extended type Myristicaceae, Eupomatia , and within several other taxa). is ancestral, truncation of the apex occurred an uncertain num- The intriguing possibility that inner staminodes ( 46 ) might ber of times in four near-basal lines (Hydatellaceae, Cabom- be a primitive feature in angiosperms ( Endress, 1984 ; Dono- baceae, Schisandraceae plus Illicium , and Sarcandra ) and in ghue and Doyle, 1989 ) is not borne out: inferred relationships mesangiosperms. With the D& E backbone a truncated apex is imply that inner staminodes originated independently in Aus- reconstructed as basic for the monocot-magnoliid clade, and in trobaileya and in Magnoliales and Laurales. In the latter groups, all trees it is ancestral in magnoliids. As a result, the extended because we scored Myristicaceae as unknown, since they have apex of the classic laminar stamens of Magnoliales is a second- highly modifi ed male fl owers with a central columnar androe- arily derived feature that unites Galbulimima , Degeneria , cium, it is equally parsimonious to assume that inner stami- Eupomatia , and Annonaceae, and in Laurales the same is true noides arose once, followed by loss in Magnoliaceae, or twice, in for Calycanthaceae. The situation is confused in eudicots: it is Laurales and the clade consisting of Galbulimima , Degeneria , equivocal whether the extended apex of Euptelea , Nelumbo , January 2009] Endress and Doyle — Ancestral flowers 39

Fig. 7. (A) D& E tree, showing inferred evolution of form of stamen base (character 48). (B) Inferred evolution of orientation of anther dehiscence (character 53). Abbreviations as in Fig. 2 . 40 American Journal of Botany [Vol. 96 and Buxaceae is ancestral or derived. A peltate apex originated aceae, or separately in the atherosperm and Lauraceae-Hernandi- independently in Nuphar and Platanus (and within Annon- aceae clades. aceae: Doyle et al., 2000; Scharaschkin and Doyle, 2006 ; see In Magnoliales embedded pollen sacs, inner staminodes, also Endress, 2008b ). food bodies, extended connective apex, and H-dehiscence ap- Embedded pollen sacs ( 51 ) are characteristic of some lami- pear to have evolved in the context of beetle pollination, so it is nar stamens, as in Degeneria , but they also occur in taxa with interesting that some of these advances also occurred in Caly- fi lamentous stamens, such as the Piperaceae-Saururaceae clade, canthaceae, another beetle-pollinated group, as early empha- and some laminar stamens have protruding pollen sacs, as il- sized by Grant (1950) . lustrated most graphically by Austrobaileya . Protruding pollen sacs are inferred to be ancestral, as in Amborella and Austrobai- Gynoecium— We have not recognized separate characters leyales, and embedded pollen sacs were derived several times, for carpel phyllotaxis and merism because these features are often uniting important groups: Nymphaeaceae, Chloranthaceae usually correlated with those of the androecium. The most con- (reversed in Chloranthus ) and Ceratophyllum (if these are re- spicuous deviation from this correlation is presence of one car- lated), Piperaceae-Saururaceae, Magnoliales other than Myris- pel ( 74), which we contrast with more than one. More than one ticaceae, once or twice in Laurales (in the “ atherosperm ” clade carpel is reconstructed as ancestral; reduction to one occurred consisting of Atherospermataceae, Gomortega , and Sipar- independently in Hydatellaceae, Trimenia , Myristicaceae, De- unaceae and the Lauraceae-Hernandiaceae clade), and Trocho- generia , the Lauraceae-Hernandiaceae clade, Berberidaceae, dendraceae. Reduction to two microsporangia ( 52 ) occurred Proteaceae, and within several groups. In the D & E trees, reduc- once (with reversals) or more times in Laurales— in the athero- tion to one carpel is an important synapomorphy of Ceratophyl- sperm clade, Lauraceae (where the ancestral state is unclear: lum and Chloranthaceae. Rohwer and Rudolph, 2005), and Hernandiaceae, nearly coincid- One of the most signifi cant results of the molecular rooting ing with the shift to embedded pollen sacs — and in Circaeaster . of angiosperms was its implication that the ancestral carpel was Orientation of dehiscence (microsporangium position) ( 53 ) not the conduplicate or plicate type of Magnoliales and Winter- is one of the most homoplastic fl oral characters (consistency aceae, similar to a leaf folded down the middle (Bailey and index, C. I. = 0.09), but it shows some noteworthy patterns (Fig. Swamy, 1951 ), but rather the ascidiate type, which grows up as 7B ). Problems in scoring of unistaminate fl owers are discussed a cup or tube as the result of a meristematic cross-zone between in Appendix 1. In Doyle and Endress (2000) , introrse dehis- the primordium margins, and was sealed not by postgenital cence (adaxial microsporangia), as in Amborella , Nymphae- fusion but by secretion in the resulting canal ( Endress and aceae, and most Austrobaileyales, was ancestral, but with the Igersheim, 2000a). Earlier, margins of some plicate carpels had addition of Hydatellaceae, which are latrorse, the ancestral ori- been described as unsealed, so that pollen tubes grew to the entation is equivocal (introrse or latrorse) in the D& E trees ovules among stigmatic hairs, but in fact they are sealed by ( Fig. 7B ). However, with the J/M backbone, introrse is still re- postgenital fusion of the immature epidermises ( Igersheim and constructed as ancestral, because the latrorse Chloranthaceae Endress, 1997). Because these topics were discussed in detail in are further from the base of the tree. The ancestral state for me- Doyle and Endress (2000) and Endress and Igersheim (2000a), sangiosperms is equivocal with all trees, but by the base of the we consider them more briefl y here, except where the two back- magnoliids dehiscence appears to have become extrorse. These bone topologies have different implications. Our conclusions results, together with those on stamen base (48), are therefore are not affected by addition of Archaefructus , in which we con- consistent with a scenario in which the introrse laminar stamens servatively scored these characters as unknown, because they of the fi rst angiosperms fi rst became more fi lamentous, and are often impossible to evaluate in the absence of developmen- then these stamens were secondarily expanded with a new, tal or anatomical data ( Endress, 2005 ). abaxial microsporangium position in magnoliids. Reversals to In all trees, the ancestral carpel form ( 75) is ascidiate. With introrse occurred in Magnolioideae, Eupomatia , and Laurales, the D& E backbone (Fig. 8A), origin of the plicate carpel is an where introrse is a synapomorphy of groups other than Caly- important synapomorphy of mesangiosperms other than Chlo- canthaceae (with reversals in Hortonia and Gyrocarpoideae and ranthaceae and Ceratophyllum, with reversals to ascidiate in much plasticity in Lauraceae, often within the same fl ower). Mollinedioideae, Circaeaster, Berberidaceae, and Nelumbo . Latrorse is widespread in eudicots, perhaps functionally related The intermediate type, with both ascidiate and plicate zones be- to the narrow fi lament of most groups, but it is equivocal as a low the stigma and the ovule(s) attached to the ascidiate zone, synapomorphy of the clade. evolved from ascidiate in Barclaya and Illicium, but also from Anther dehiscence (54 ) by longitudinal slits is clearly ances- plicate in Myristicaceae, Laurales other than Calycanthaceae, tral. Branching of the ends of the slit, resulting in “ H-valvate ” Acorus , and Euptelea . In contrast, with the J/M backbone ( Fig. dehiscence, occurred in Nuphar , Monimioideae (Laurales), 8B ), with Chloranthaceae and Ceratophyllum nested at differ- Euptelea , Platanus , within Calycanthoideae (Sinocalycanthus ; ent positions in mesangiosperms, scenarios in mesangiosperms Staedler et al., 2007), and as a synapomorphy of Sarcandra and are equivocal. Either the ascidiate carpels of Chloranthaceae Chloranthus , the Galbulimima -Annonaceae clade (Magnoliales), and Ceratophyllum are primitive and plicate carpels originated and Trochodendraceae. In Nymphaeales, Chloranthaceae, and separately in eudicots, monocots, and magnoliids, or plicate Magnoliales, it appears to have evolved after embedded pollen carpels originated at the base of mesangiosperms and reversed sacs. This feature is also known from a number of unplaced Cre- twice to ascidiate in Chloranthaceae and Ceratophyllum . In taceous fossils ( Friis et al., 1991 , 2006 ) and the Late Cretaceous monocots, the intermediate carpels of Acorus may or may not calycanthoid fl ower Jerseyanthus (Crepet et al., 2005). Dehis- be evolutionarily intermediate between ascidiate and plicate, cence by apically hinged fl aps is a distinctive feature of many and the ascidiate carpels of some Araceae may be either primi- Laurales, but like basal glands, which have a partially overlap- tive or derived. ping distribution, its history is equivocal: it may have arisen after As already noted, we split modes of carpel sealing into two divergence of Calycanthaceae, with a reversal within Monimi- characters. Scenarios for origin of postgenital fusion of the January 2009] Endress and Doyle — Ancestral flowers 41

Fig. 8. (A) D& E tree, showing inferred evolution of carpel form (character 75; state 1 = intermediate, with ovule(s) on ascidiate zone). (B) J/M tree, showing different reconstruction of evolution of same character. Abbreviations as in Fig. 2 . 42 American Journal of Botany [Vol. 96 carpel margins (76 ) in mesangiosperms are similar to those for reversal in the Piperales-Canellales clade. In any case, the long origin of the plicate carpel, differing with the D & E and J/M stigmatic crest of Degeneria , often interpreted as a primitive trees. However, the two characters are not redundant, because feature, appears to be a reversal. complete fusion also arose at the base of Nymphaeaceae, when Stigmatic protuberances ( 81 ), found in Amborella , may be the carpels were still fully ascidiate, and its history within mag- ancestral if Amborella is basal in angiosperms, but not if Ambo- noliids, monocots, and eudicots was different. Laurales other rella and Nymphaeales form a clade. Other occurrences origi- than Calycanthaceae shifted to intermediate carpels but have nated independently, in Trimenia , Chloranthaceae (with a loss complete or partial postgenital fusion, and complete fusion may in Sarcandra ), Idiospermum , and Hydrastis plus Glaucidium . have persisted into Lauraceae and Hernandiaceae. Euptelea and Stigmatic papillae ( 82) with either a pluricellular ( Amborella , Acorus also have intermediate carpels but complete postgenital Hydatellaceae, Barclaya , Nymphaeoideae) or unicellular emer- fusion. Many monocots (aquatic Alismatales, the Melanthi- gent portion may be ancestral, but the unicellular state was es- aceae-Dioscoreales clade) are plicate but have only partial post- tablished in the common ancestor of Austrobaileyales and genital fusion, and some Ranunculales are plicate but have no mesangiosperms, followed by scattered origins of pluricellular or partial postgenital fusion. As a result, with the J/M backbone papillae in Trimenia , Asaroideae, Degeneria , Eupomatia , and ( Fig. 9A ), it is equally parsimonious to assume that lack of post- Butomus, and losses of papillae in Ceratophyllum , Sarcandra genital fusion persisted well into both monocots and eudicots, plus Chloranthus , Berberidaceae, and Hydrastis . even though they were plicate, or that there were reversals of Formation of an extragynoecial compitum ( 83 ), where con- postgenital fusion within these groups. Secretion in the carpels tact between stigmas allows pollen tubes to grow to more than ( 77) persisted well into groups with plicate carpels and post- one carpel, appears to be ancestral in angiosperms and was re- genital fusion, being lost once or twice in Canellales and Piper- tained through Austrobaileyales, with a loss in Cabombaceae. ales; in Degeneria , Eupomatia , Calycanthaceae, Gomortega , With the D& E backbone (Fig. 10A), this feature is lost near the and the Lauraceae-Hernandiaceae clade; and an uncertain num- base of the mesangiosperms (the state in Chloranthaceae is un- ber of times in eudicots, always either coincident with or subse- defi ned because they have only one carpel), followed by reap- quent to postgenital fusion. However, secretion was retained in pearances in Magnoliales and Laurales (once with a loss in monocots. Magnoliaceae, or independently in the Galbulimima -Annon- A single cell layer of pollen tube transmission tissue ( 78 ) aceae clade and in Laurales) and (once or twice) in Lardizabal- originated repeatedly: in Austrobaileyales (once or twice: it is aceae and Menispermaceae. With the J/M backbone, both this absent in Trimenia), Asaroideae (Piperales), Canellales, the scenario and one in which an extragynoecial compitum per- Galbulimima -Annonaceae clade (Magnoliales), Hortonia sisted into Magnoliales and Laurales, with parallel losses in the (Monimiaceae), monocots, and three times in eudicots (Lard- monocot-eudicot and Piperales-Canellales clades, are equally izabalaceae, Berberidaceae-Ranunculaceae, and the clade of parsimonious. Actual fusion of carpels (84 ; Fig. 10B ) occurred Proteales, Trochodendraceae, and Buxaceae). However, it several times by two different routes. Eusyncarpy, with carpels seems stable within these groups (except possibly Austrobai- fused at the center of the gynoecium, often resulting in axile leyales). This character needs more study, since the “ differenti- placentation, evolved independently in Nymphaeaceae, Aristo- ated ” state includes more than one type of cell differentiation. lochiaceae, monocots, and the Trochodendraceae-Buxaceae Most signifi cant is a third state, multilayered transmission tis- (and gunnerid) clade. The topology in monocots implies that sue, which is one of several morphological synapomorphies of the free carpels of Alismatales other than Araceae are not prim- Lauraceae and Hernandiaceae. itive but rather secondarily derived from united carpels, as con- The most homoplastic character is formation of a style ( 79 ; cluded by Chen et al. (2004). However, the situation in the C. I. = 0.05 – 0.06). The reconstructed ancestral state is either Piperales-Canellales clade is confused: Aristolochiaceae are lack of a style (i.e., sessile stigma) or equivocal, depending on eusyncarpous (or paracarpous in some presumably derived the backbone tree, the arrangement of Amborella and Nympha- Aristolochia species), but Piperaceae-Saururaceae, Canel- eales, and addition of Archaefructus, which has a style (see laceae, and Takhtajania in the Winteraceae ( Endress et al., Table 1). The basic state in mesangiosperms is presence of a 2000 ) are paracarpous, with carpels fused into a unilocular style with the D & M backbone, followed by many losses (e.g., ovary with parietal placentation, and other Winteraceae and one or two in Magnoliales), but equivocal with the J/M back- Lactoris are apocarpous. One scenario is that apocarpy was an- bone — a style may have originated once or many times. How- cestral; paracarpy originated independently in the Piperaceae- ever, in some clades a style seems to have persisted once Saururaceae clade, Takhtajania , and Canellaceae; and formed, as in most of the Laurales, Alismatales other than Ar- eusyncarpy evolved from apocarpy in Aristolochiaceae. In aceae, the Melanthiaceae-Dioscoreales clade (petrosaviids), other scenarios, paracarpy was ancestral and reversed to and the Buxaceae-Trochodendraceae clade (probably including apocarpy in Lactoris and Winteraceae. In any case, paracarpy gunnerids). Stigma extension ( 80 ) is less homoplastic ( Fig. evolved independently in Papaveraceae. 9B ): a stigma extending more than halfway down the style- Several minor modifi cations involve the carpel surface. In- stigma zone is reconstructed as ancestral, and it did not become trusive oil cells (85 ) visible at the carpel surface (scored only in restricted to the apex until within mesangiosperms. In the J/M taxa with mesophyll oil cells) originated independently in Aus- trees, where Chloranthaceae (with an extended stigma) are trobaileyales (Schisandraceae, Illicium , and possibly Trimenia , linked with magnoliids, this occurred four times, in the Magno- which is mixed), Sarcandra plus Chloranthus , the Piperaceae- liales-Laurales clade, monocots, Ranunculales other than Eupt- Saururaceae clade, and once or twice in Monimiaceae (Horto- elea (Papaveraceae scored as unknown because the stigma is nia and Mollinedioideae). Long unicellular hairs ( 86) on and/ modifi ed by syncarpy; reversed in Berberidaceae), and Pro- or between the carpels may be a synapomorphy of Laurales, but teaceae; but in the D & E trees ( Fig. 9B ), where Chloranthaceae this is equivocal because they are absent in Gomortega and Si- are basal in mesangiosperms, restriction of the stigma may or parunaceae; similar hairs also evolved in Hydrastis and within may not be homologous in monocots and magnoliids, with a several taxa. Short curved hairs ( 87) with a long apical cell January 2009] Endress and Doyle — Ancestral flowers 43

Fig. 9. (A) J/M tree, showing inferred evolution of postgenital fusion of carpel margins (character 76). (B) D& E tree, inferred evolution of stigma extension (character 80). Abbreviations as in Fig. 2 . 44 American Journal of Botany [Vol. 96

(Endress, 2001) are found in Amborella , Nymphaeales (except marginal placentation in Nymphaeales (the state in Hydatel- Nuphar ), and sometimes Trimenia and may be ancestral in an- laceae is unknown, because the fl owers lack orientation marks) giosperms. Abaxial nectaries (88 ) on the backs of the carpels and Butomus (and presumably related Alismatales). The ances- are a synapomorphy of Buxaceae and Trochodendraceae. A tral ovule direction ( 92 ; Fig. 12 ) is reconstructed as pendent, widespread feature in monocots is septal nectaries ( 89 ) be- both in basal groups with one apical ovule, such as Amborella tween the fused carpels, seen in our data set in Dioscoreaceae and Hydatellaceae, and in multiovulate Nymphaeales. Aus- and some Nartheciaceae. It has been suggested that lateral nec- trobaileyales show one or two shifts to horizontal, in Austrobai- taries on the free carpels of some Alismatales (represented by leya and Schisandraceae, and one to ascendent, in Illicium . With Tofi eldiaceae and Butomus) may be homologous (Daumann, the D & E tree, pendent persists to Chloranthaceae and into basal 1970; Igersheim et al., 2001), and we scored them as the same eudicots, but in the monocot-magnoliid clade ovule direction state, but they originate independently on the trees. may either remain pendent or shift to ascendent. With the J/M Consideration of the last two characters and others treated tree, because Chloranthaceae and Ceratophyllum are nested in earlier indicates that nectaries originated twice on the adaxial mesangiosperms, the pendent state unambiguously persists up to side of the inner perianth parts (37), in Cabomba and Ranuncu- Acorus in the monocots. With both trees, a shift to the ascendent lales; once or twice on the stamen bases (49) in Laurales; once state occurs within Ranunculales (Menispermaceae, Berberi- on the backs of the carpels (88) within eudicots; and two or daceae, and Ranunculaceae). The basic state in monocots other more times on the sides of the carpels (89) in monocots. Other than Acorus is ascendent; this may be either ancestral for mono- types that we did not include because they differ morphologi- cots (D & E) or derived from pendent (J/M). The ancestral state in cally and appear to be autapomorphic are abaxial nectaries on magnoliids is entirely unresolved, but horizontal is basic in Pip- the petals of Nuphar (Hiepko, 1965; Endress, 2008a) and disc- erales-Canellales and Magnoliales other than Myristicaceae like nectaries of uncertain morphological nature in Proteaceae (which have one basal, ascendent ovule). Ascendent is ancestral (Douglas, 1995) and Sabiaceae (not included in this data set; in Laurales, reverting to pendent in Gomortega and in the clade Ronse De Craene and Wanntorp, 2008 ). These results imply consisting of Monimiaceae, Lauraceae, and Hernandiaceae. that nectar secretion itself arose independently at these different Inferences on the ancestral ovule curvature (93 ) depend on sites: even if all cases of nectar secretion were treated as a state interpretation of Amborella , described as anatropous by Bai- of one character, none of the different types of nectaries would ley and Swamy (1948), orthotropous by Endress and Iger- be inferred to be homologous. sheim (2000b) and Yamada et al. (2001) , and hemianatropous In our previous analysis (Doyle and Endress, 2000), the an- (= hemitropous) by Tobe et al. (2000) . Based on the illustra- cestral ovule number ( 90 ) was equivocal: either one, as in Am- tions of both Endress and Igersheim (2000b) and Tobe et al. borella , Trimenia , Illicium , and Chloranthaceae, or more than (2000) , the funicle is attached to one side of the base of the two, as in most core Nymphaeales and Austrobaileya. Now, be- ovule, not halfway along the side of the ovule, as in the typical cause of the addition of Hydatellaceae, which are uniovulate, hemitropous condition. The asymmetry of the ovule base may our data imply that one ovule is ancestral when only Recent be a consequence of the “ apical ” position of the ovule, which taxa are considered ( Fig. 11A ) and that this number was re- is actually attached to the adaxial cross zone; in our experi- tained up to Trimenia and Illicium in the Austrobaileyales and ence, a truly symmetrical base is restricted to taxa with either to Chloranthaceae (plus Ceratophyllum in D& E trees). How- basal ovules or apical ovules in a spacious locule (e.g., Acorus ; ever, if Archaefructus , which has several ovules per carpel, is Buzgo and Endress, 2000 ). Because the Amborella condition linked with Hydatellaceae, the ancestral state is still equivocal seems closer to typical orthotropous than to anatropous, we (Fig. 11B). Scenarios in mesangiosperms vary with the back- have included it in the orthotropous state. With this scoring, bone tree. With the D & E trees ( Fig. 11 ), where Ceratophyllum the ancestral ovule curvature is equivocal if Amborella is sis- and Chloranthaceae form a clade at the base of the mesangio- ter to all other angiosperms. However, the fact that the outer sperms, the basic ovule number in the remaining mesangio- integument is asymmetric during development in both Ambo- sperms is entirely equivocal, and the uniovulate condition in rella and Chloranthus (Yamada et al., 2001) suggests that magnoliid groups such as Myristicaceae, Galbulimima , and orthotropous was derived from anatropous ( Endress and Laurales (other than Calycanthaceae, which have two ovules) Igersheim, 2000b ). This is the most parsimonious hypothesis may be either a retention from the fi rst angiosperms or the result if Amborella and Nymphaeales form a clade. It is also favored of reduction. However, the basic number in the Piperales- if the bitegmic ovule is homologous with the cupule of Cayto- Canellales clade and in monocots is more than two, and the nia (Gaussen, 1946; Stebbins, 1974; Doyle, 1978), as indi- basic number in eudicots is either two or more. In contrast, with cated by some cladistic analyses of seed plants (Crane, 1985; the J/M backbone, where Ceratophyllum and Chloranthaceae Doyle and Donoghue, 1986 ; Doyle, 2006 , 2008; Hilton and are nested at different points within the mesangiosperms, the Bateman, 2006 ). Orthotropous ovules evolved several other uniovulate condition is retained from the base of the angio- times, sometimes uniting clades: in Barclaya (Nymphae- sperms to these groups (unless Archaefructus is included, in aceae), Chloranthaceae and Ceratophyllum (a clade in D & E which case the uniovulate condition in Ceratophyllum may be trees), Piperaceae and Saururaceae, Gomortega , Acorus , Cir- either primitive or secondary) and into Magnoliales and Lau- caeaster, and the Platanus -Proteaceae clade. rales. This scenario would imply there were increases in ovule number in the Piperales-Canellales clade, Magnoliaceae, the Summary of character discussion— Our results ( Table 1 ) Galbulimima-Annonaceae clade, Calycanthaceae, monocots, imply that the ancestral angiosperm fl ower had more than two and eudicots. Secondary reduction to one ovule occurred in whorls (or series) of tepals, more than two whorls (series) of Piperaceae and Nelumbo . stamens, probably with adaxial microsporangia (introrse), and Laminar placentation ( 91; including “ dorsal ” in Brasenia , several ascidiate carpels that were sealed by secretion rather actually on the carpel midrib), often noted as a similarity of than postgenital fusion, most likely with one pendent bitegmic Nymphaeales and Alismatales, was independently derived from ovule, which was probably anatropous. These fl owers were January 2009] Endress and Doyle — Ancestral flowers 45

Fig. 10. (A) D& E tree, showing inferred evolution of an extragynoecial compitum (character 83). (B) Inferred evolution of carpel fusion (syncarpy; character 84). Abbreviations as in Fig. 2 . 46 American Journal of Botany [Vol. 96 borne either in racemes (which some authors might call shoots whorls (or series) of perianth parts, several free carpels, and with axillary solitary fl owers) or in botryoids. Perianth and an- probably bisexuality. However, possession of more than two droecium phyllotaxis is uncertain, but if parts were whorled whorls (or series) of stamens may be a secondary increase from they were trimerous. The most striking uncertainty is whether an intermediate stage with two whorls near the base of the me- the ancestral fl ower was bisexual or unisexual. This is an area sangiosperms. The laminar form of many magnoliid stamens where comparative studies on the genetic control of develop- may also be a reversal from more fi lamentous stamens in earlier ment and better understanding of fossil diversity could be most mesangiosperms, and the abaxial position of the microsporan- interesting. The clearest effect of considering Archaefructus gia in many magnoliids, which may refl ect this secondary ex- concerns ovule number— whereas analysis of Recent taxa alone pansion, is more defi nitely derived. Whether the fi rst implies that one ovule was ancestral, the ancestral state be- angiosperms had spiral or whorled fl oral phyllotaxis, the spiral comes ambiguous (either one or more than two) if Archaefruc- perianth of some magnoliids appears to be derived from a tus is linked with Hydatellaceae. whorled perianth lower in the mesangiosperms, and the same The “ explosive ” radiation of angiosperms appears to have may be true for the androecium. Most notably, the plicate car- begun with the origin of the mesangiosperm clade, after the ori- pels often illustrated in textbooks as primitive are instead de- gin of crown group angiosperms and divergence of the more rived, as is the elongate “ strobilar ” receptacle of Magnoliaceae. basal ANITA lines (cf. Moore et al., 2007). It should be noted Evidence is also strong that the solitary fl owers of many mag- that the beginning of this radiation, corresponding to the initial noliids are derived. Many of these derived features may be re- splitting of the main mesangiosperm lines, must have predated lated to a general increase in fl ower size and specialization for the radiation of angiosperms observed in the latter half of the beetle pollination, like the inner staminodes of many Magno- Early Cretaceous fossil record (Barremian through Albian), liales and Laurales. which involved diversifi cation within the magnoliid, eudicot, and monocot clades (Doyle and Hickey, 1976; Hughes, 1994; Floral groundplan of simple fl oral structure in near-basal Doyle, 2001 ; Friis et al., 2006 ; Doyle and Endress, 2007 ). There angiosperms— The drastically simple reproductive structures is no unequivocal fl oral synapomorphy at this point in the tree of several extant basal angiosperms and Early Cretaceous fos- that might be interpreted as a key innovation responsible for sils have provoked much recent discusion. In Ceratophyllum , this radiation (cf. Cantino et al., 2007). Most mesangiosperms fl owers are unisexual; female fl owers are unicarpellate, with an differ from the more basal lines in having plicate rather than ascidiate carpel. The structures commonly interpreted as multi- ascidiate carpels, sealed by postgenital fusion rather than secre- staminate male fl owers (Endress, 1994b; Iwamoto et al., 2003) tion. However, Chloranthaceae have ascidiate carpels that are are more likely infl orescences of unistaminate fl owers without notably similar to those of more basal groups (Endress, 1987b, subtending bracts ( Endress, 2004 ), as we argued earlier. This 2001 ; Endress and Igersheim, 2000a). If the Doyle and Endress interpretation becomes even more plausible if a relationship of (2000) arrangement based on combined molecular and morpho- Ceratophyllum and Chloranthaceae is envisaged ( Duvall et al., logical data is correct and Chloranthaceae (with or without 2006; Mathews, 2006; Qiu et al., 2006), as supported by our Ceratophyllum) are basal in mesangiosperms, plicate carpels analysis. The male structures of Hedyosmum are also infl ores- originated at the next node; if Chloranthaceae are nested within cences of unistaminate, perianthless fl owers without subtend- mesangiosperms ( Jansen et al., 2007 ; Moore et al., 2007 ), pli- ing bracts ( Endress, 1987a ). Although Leroy (1983) interpreted cate carpels may have originated either at the base of mesangio- these male shoots as fl owers, the infl orescence interpretation is sperms (but soon reversed) or several times within the clade. more plausible based on comparison with male infl orescences Furthermore, the fact that Chloranthaceae are one of the most in Ascarina , in which each stamen (or two to three stamens) is prominent recognizable groups in the Early Cretaceous fossil (are) subtended by a small subtending bract. Male shoots simi- record (Friis et al., 1994, 2006 ; Eklund et al., 2004; Feild et al., lar to those in Hedyosmum are also known from the Early Cre- 2004 ) suggests they were part of any accelerated radiation. An taceous ( Friis et al., 1994 , 2006 ). apomorphy more closely tied to the base of the mesangiosperms Hydatellaceae and Archaefructus ( Sun et al., 2002 ) exhibit a is origin of the typical eight-nucleate female gametophyte and similar pattern of very simple fl owers. In Hydatellaceae, sev- resultant formation of triploid rather than diploid endosperm eral stamens are commonly surrounded by a number of ascidi- ( Friedman and Williams, 2003 , 2004 ; Friedman, 2008 ; Rudall ate carpels, and these by two or more bracts (Rudall et al., et al., 2008 ), but the history of this character is ambiguous be- 2007). The simplest interpretation is that the fl owers are uni- cause Amborella has a nine-nucleate female gametophyte sexual, unicarpellate or unistaminate, and perianthless ( Friedman, 2006 ). (Hamann, 1975, 1976 , 1998 ; Rudall et al., 2007). In Archae- The fact that molecular data fi rmly nest Magnoliidae (in the fructus , we interpret the fl owers as unisexual, 1 – 2-carpellate or restricted monophyletic sense of Cantino et al., 2007) well usually 2-staminate, and perianthless (Friis et al., 2003). Some within the angiosperms calls into question the traditional use of authors have suggested that such structures may represent a magnoliid groups such as Magnoliales and Winteraceae as prefl oral state ( Sun et al., 2002 ; Friis and Crane, 2007 ) or may models for the original angiosperm fl ower (e.g., Cronquist, be a result of secondary dissolution of the fl ower – infl orescence 1968 ; Takhtajan, 1969 ). However, fl owers of these groups are boundary due to loss of fl oral identity (Rudall et al., 2007). Sun more like our present reconstruction of the ancestral fl ower than et al. (2002) speculated that the fertile structures of Archaefruc- some that were being discussed before the molecular rooting— tus might represent an evolutionary stage at which the genetic e.g., the simple fl owers of Chloranthaceae, suggested as one of programs for fl ower and infl orescence formation were not yet several alternative prototypes by Endress (1986a) and found to strictly separated. be basal in some morphological cladistic analyses ( Nixon et al., Our inferences on infl orescence, perianth, and androecium 1994; Hickey and Taylor, 1996). According to our analysis, evolution do not support the suggestion that these taxa represent many putatively “ primitive ” magnoliid features were indeed a prefl oral state. Although developmental studies show that a retained from the fi rst angiosperms, such as more than two perianth is completely missing in Hydatellaceae, Ceratophyl- January 2009] Endress and Doyle — Ancestral flowers 47

Fig. 11. (A) D& E tree, showing inferred evolution of number of ovules per carpel (character 90). (B) D& E tree after addition of Archaefructus , show- ing different reconstruction of evolution of same character. Hernandioideae and Gyrocarpoideae combined as Hernandiaceae. Abbreviations as in Fig. 2 . 48 American Journal of Botany [Vol. 96 lum, and most Chloranthaceae (except female fl owers of Hedyos- weight than many others and a reason to exercise caution in ac- mum), and not even present as rudiments in early developmental cepting relationships based on them. Physical conditions are stages (Endress, 1987b, 1994b ; Kong et al., 2002; Iwamoto et very different in the water and in the air, and the evolutionary al., 2003 ; Rudall et al., 2007 ), our results imply that the simple transition from the air to the water has a profound impact on fl oral structure of these groups and Archaefructus is a result of plant structure and fl ower biology (Sculthorpe, 1967; Vogel, reduction (i.e., decrease in organ number, loss of a perianth, and 1996; Voesenek et al., 2006). A morphological interpretation of probably loss of bisexuality) of more “ complete ” ancestral fl ow- submerged fl owers should take into account these differences. ers with a perianth and several stamens and carpels. On parsi- Among angiosperms systematic surveys show many in- mony grounds, it is equivocal whether the ancestral fl ower was stances of evolution from the land to the water ( Cook, 1999 ), bisexual, but we suspect it was, for the reasons discussed. and shifts from pollination in the air to under the water (or on On phylogenetic grounds, it is less easy to eliminate the hy- its surface) also occurred a number of times, whereas there are pothesis that fl oral reduction in these groups occurred not by no clear reversals. Based on our survey, there is clearly a gen- gradual loss of parts but by loss of fl oral identity, one of three eral tendency for underwater-fl owering plants to evolve simple, possibilities discussed by Rudall et al. (2007) . However, if Ar- unisexual, perianthless, and unistaminate or unicarpellate fl ow- chaefructus is related to either Hydatellaceae or Ceratophyl- ers. This reduction trend is especially conspicuous in near-basal lum , the fact that its fl owers had usually two stamens and one or monocots (Alismatales), but it is also recognizable (usually in two carpels may support a reduction scenario. Loss of fl oral less extreme form) in other angiosperms. In Alismatales there is organs may have been easier in more basal angiosperms be- a broad diversity of plants with underwater fl owering, diverse cause they had less fl oral synorganization than more derived modes of water-surface fl owering, and fl owering in the air. clades. However, we see no reason to think that this involved Phylogenetic analyses show that the taxa with submerged fl ow- loss of the basic fl oral program. A more conservative hypothe- ers are derived from taxa with aerial fl owers. That there is really sis is that the basic fl oral program was still present but the fl ow- an evolutionary trend is shown by the fact that underwater pol- ers became simpler by loss and reduction of organs, and lination did not evolve just once in Alismatales but several selection for maintenance of suffi cient reproductive output times ( Les et al., 1997 ; Chen et al., 2004 ). favored production of more fl owers per individual or per Another possibility to consider is that the underwater-fl ow- infl orescence. ering habit may have often evolved from wind pollination. A This view is supported by recent molecular developmental number of water plants have emergent infl orescences and studies on Amborella and Nymphaeales, which suggest that the wind-pollinated fl owers ( Cook, 1988 ). Wind pollination and fl oral genetic program originally found in Arabidopsis and An- underwater pollination can lead to similar reductions in fl oral tirrhinum (Coen and Meyerowitz, 1991) is present with similar structure (see discussion in Endress, 1994a). Thus features function in these basalmost extant angiosperm lines. Further- adapted to wind pollination may have been preadaptations for more, the program does not differ profoundly between Ambo- underwater pollination. This evolutionary pathway is consis- rella and Nymphaeales, in which Nuphar has been studied tent with the fact that there are genera in which wind pollina- ( Soltis et al., 2006 ). The same is true for Illicium , another mem- tion and underwater pollination co-occur, such as Groenlandia ber of the ANITA grade, and some magnoliids (Asimina , Eupo- (Potamogetonaceae) (Guo and Cook, 1990) and Callitriche matia , Liriodendron , Magnolia , Persea: Kim et al., 2005a, b ; (Plantaginaceae) ( Philbrick and Les, 2000 ). However, in gen- Chanderbali et al., 2006 ; Soltis et al., 2006, 2007 ; Buzgo et al., eral, fl oral reduction is even stronger in water-pollinated than 2007). In Chloranthus class A genes (responsible for perianth in wind-pollinated fl owers. This scenario is a real possibility identity in Arabidopsis and Antirrhinum) are present, although for Ceratophyllum if it is related to Chloranthaceae, which ap- its fl owers do not have a perianth ( Li et al., 2005 ). It appears pear to be ancestrally wind-pollinated, as in Hedyosmum and that one of the mechanisms for the early evolutionary modifi ca- Ascarina (Endress, 1987b). tion and elaboration of the fl oral developmental program was In Alismatales, there are eight families in which some or repeated events of gene duplication and sub- or neofunctional- all members have submerged fl owers (Cymodoceaceae, Hy- ization (Irish and Litt, 2005; Zahn et al., 2005; Irish, 2006; drocharitaceae, Juncaginaceae, Posidoniaceae, Potamoget- Kramer and Zimmer, 2006). This is certainly a promising track onaceae, Ruppiaceae, Zannichelliaceae, and Zosteraceae). to follow in evo-devo studies. These families are representatives of both major aquatic clades in the order ( Chase et al., 2006 ; Les et al., 2006 ). Of Extant angiosperms that fl ower under water and peculiari- these eight families, fi ve have unisexual fl owers. Six have pe- ties of their reproductive structures— Because Hydatellaceae, rianthless fl owers, some with a spathe-like envelope (vs. two Archaefructus , and Ceratophyllum are or were water plants, it with a single whorl of perianth organs). It is uncertain whether is useful to consider if and how the scenarios developed here this envelope is a modifi ed perianth or a bract. Five have uni- may be functionally related to a shift from a terrestrial to an staminate or bistaminate male fl owers (vs. three with three or aquatic habitat. For this purpose, we cursorily surveyed repro- more stamens). Seven have at least partly unicarpellate or bi- ductive morphology in all angiosperm families comprising taxa carpellate female fl owers (vs. one with three or more carpels) with underwater fl owers. Comparisons with these taxa suggest (Doty and Stone, 1966; Tomlinson, 1969, 1982 ; Cook, 1998b; that fl oral simplicity comparable to that in more basal aquatic Haynes et al., 1998a, b, e , g; Kuo and McComb, 1998a – c ; groups has frequently arisen by reduction. We emphasize that Igersheim et al., 2001 ). we are not using these correlations as evidence for one or an- In contrast, there are seven families in Alismatales without other concept of relationships or direction of evolution; rather, submerged fl owers (Alismataceae, Aponogetonaceae, Araceae, we present them as possible biological explanations for the Butomaceae, Limnocharitaceae, Scheuchzeriaceae, Tofi el- changes inferred from phylogenetic trees. However, it would be diaceae). In all of these, the fl owers are either always or some- fair to take functional correlations among reduced fl oral fea- times bisexual. All have a perianth of two whorls, six or more tures as evidence that these characters have less individual stamens, and at least three carpels, except for derived groups of January 2009] Endress and Doyle — Ancestral flowers 49

Fig. 12. D & E tree, showing inferred evolution of ovule direction (character 92). Abbreviations as in Fig. 2.

Araceae ( Kaul, 1968 ; Cook, 1998a ; Haynes et al., 1998c , d, f ; short-styled carpel with no bract-like organ; and (5) female fl ow- Mayo et al., 1998 ; van Bruggen, 1998 ; Igersheim et al., 2001 ). ers of one very long-styled carpel with no bract-like organ. A family of Alismatales that particularly well illustrates the Whether the bract-like organ is a fl oral subtending bract or a pe- evolutionary fl exibility of fl owers is Juncaginaceae. It shows not rianth part, the fl owers are extremely simple in terms of organ only an evolutionary transition from aerial to submerged fl owers numbers. and correlated morphological changes but also conspicuous Thus, although more surveys from a phylogenetic perspec- lability of fl ower forms in the group with submerged fl owers. tive would be desirable, Alismatales can serve as a model group Some Juncaginaceae have spikes of aerial fl owers (e.g., Tri- to show functional changes in reproductive structures correlated glochin), which are trimerous and bisexual, with two whorls of with the transition from aerial to submerged fl owers. Similar tepals. Lilaea, however, is a submerged water plant, also with patterns can be seen in other groups with completely or partly spikes. Phylogenetic analyses nest Lilaea within the family ( Les submerged fl owers. In Lamiales, Callitriche (Plantaginaceae) et al., 1997 ; Chen et al., 2004 ; von Mering and Kadereit, 2006 ), has unisexual, perianthless fl owers, male fl owers unistaminate, implying that its submerged habit is derived. There are fi ve dif- female fl owers with two carpels; Hydrostachys (Hydrostachy- ferent fl oral morphs within one species, all very simple, includ- aceae) has unisexual, perianthless fl owers, male fl owers uni- or ing from the top downward in a spike ( Posluszny et al., 1986 ): bistaminate, female fl owers with two carpels ( Erbar and Leins, (1) male fl owers, unistaminate, associated with a bract-like or- 2003, 2004 ). The two genera have the most reduced fl owers in gan, which could be a fl oral subtending bract or a tepal (inter- Lamiales, which usually have bisexual fl owers with a perianth preted as a tepal by Posluszny et al., 1986); (2) bisexual fl owers, of two whorls and at least two stamens. In Malpighiales, some unistaminate and with a single carpel, associated with a bract- Podostemaceae have submerged (cleistogamous) fl owers, many like organ; (3) female fl owers of a single short-styled carpel, as- perianthless and uni- or bistaminate (Cook and Rutishauser, sociated with a bract-like organ; (4) female fl owers of one 2007); some species of Bergia and Elatine (Elatinaceae) have 50 American Journal of Botany [Vol. 96 submerged, very small, di- or trimerous fl owers (Cook, 1990). position of Ceratophyllum underline the dangers of associating In Myrtales, some species of Rotala (Lythraceae) have sub- it with a particular extant group. More secure conclusions on its merged fl owers, some apetalous, some unistaminate ( Cook, affi nities may require recognition of other fossils that link it 1979 ). In all these groups, reduction (or loss) of the perianth and with one rather than another living taxon. Whether Archaefructus reduction of stamens are obvious from outgroup comparison. affects ideas about the fi rst fl ower, it reveals important early However, the tendency for reduction in carpel number inferred trends in fl oral evolution and the early ecological radiation of in Alismatales is not evident in the eudicot examples, perhaps angiosperms (cf. Feild et al., 2004 ). Especially if it is related to because of more intimate synorganization of the carpels. the Albian genera Vitiphyllum and Caspiocarpus ( Friis et al., In addition to reduction of the perianth, another apparent 2003), which had similar but less fi nely dissected leaves, it rep- trend in water plants is reduction or loss of the fl oral subtending resents an important trend for invasion of Early Cretaceous bract. In underwater-fl owering Alismatales, for example, the aquatic ecosystems by angiosperms (cf. Mart í n-Closas, 2003), bract is often absent in female fl owers (and rarely in male fl ow- represented today only by Hydatellaceae, core Nymphaeales, ers) of Najas (Hydrocharitaceae; Haynes et al., 1998a) and in and probably Ceratophyllum . some fl owers of Lilaea (Juncaginaceae) (Posluszny et al., 1986) (discussed earlier). However, this trend is also seen in taxa with LITERATURE CITED pollination above the water, including not only Acorus and var- ious Alismatales (Aponogeton , Araceae, Juncaginaceae, Pota- Aboy , H. E. 1936 . A study of the anatomy and morphology of Ceratophyllum mogetonaceae), but also some Nymphaeaceae (Nuphar , demersum . M.S. thesis, Cornell University, Ithaca, New York, USA. Nymphaea). This observation is of interest in view of the fact Bailey , I. W. , and B. G. L. Swamy . 1948 . Amborella trichopoda Baill., that the putative fl owers of Archaefructus do not have a subten- a new morphological type of vesselless dicotyledon. Journal of the Arnold Arboretum 29 : 245 – 254 . ding bract ( Sun et al., 2002 ; Friis et al., 2003 ). An ecological Bailey , I. W. , and B. G. L. Swamy . 1951 . The conduplicate carpel of explanation for this trend is that in water plants, whether they dicotyledons and its initial trends of specialization. American Journal fl ower in the air or in the water, the fl owers begin their develop- of Botany 38 : 373 – 379 . ment in the water and therefore do not need protection against Barkman , T. J. , G. Chenery , J. R. McNeal , J. Lyons-Weiler , W. desiccation. In nonwater plants the fl oral subtending bract pro- J. Elisens , G. Moore , A. D. Wolfe , and C. W. dePamphilis . vides such protection for the delicate young fl oral organs before 2000 . Independent and combined analyses of sequences from all the outer perianth organs are differentiated enough to take over three genomic compartments converge on the root of fl owering plant this function ( Endress, 1994a ). phylogeny. Proceedings of the National Academy of Sciences, USA Groups with submerged fl owers often have bracts below the 97 : 13166 – 13171 . Bateman , R. M. , J. Hilton , and P. J. Rudall . 2006 . Morphological and whole infl orescence, even when they lack fl oral subtending molecular phylogenetic context of the angiosperms: Contrasting the bracts, as in Hydatellaceae and some Alismatales. The apparent “ top-down ” and “ bottom-up ” approaches used to infer the likely char- absence of infl orescence bracts in Archaefructus ( Sun et al., acteristics of the fi rst fl owers. Journal of Experimental Botany 57 : 2002 ; Friis et al., 2003 ) might be considered evidence against 3471 – 3503 . the hypothesis that it was adapted to an underwater fl owering Baum , D. A. , and L. C. Hileman . 2006 . A developmental genetic model habit. However, when there are no fl oral subtending bracts that for the origin of the fl ower. In C. Ainsworth [ed.], Flowering and its individually protect youngest fl oral stages, infl orescences may manipulation, 3 – 27. Blackwell, Sheffi eld, UK. have some protection by more basal leaves. In Archaefructus , Borsch , T. , K. W. Hilu , J. H. Wiersema , C. L ö hne , W. Barthlott , infl orescences were enclosed by leaves in bud, as shown in the and V. Wilde . 2007 . Phylogeny of Nymphaea (Nymphaeaceae): type specimen of A. eofl ora (Figs. 2, 26 in Ji et al., 2004), which Evidence from substitutions and microstructural changes in the chlo- roplast trnT-trnF region. International Journal of Plant Sciences has younger stages of reproductive parts than A. sinensis in Sun 168 : 639 – 671 . et al. (2002) . In Cabombaceae, young infl orescences are en- Burleigh , J. G. , and S. Mathews . 2004 . Phylogenetic signal in nucleotide closed by regular leaves below the infl orescence and fl oral sub- data from seed plants: Implications for resolving the seed plant tree of tending leaves, and the same is true of some Alismatales. In the life. American Journal of Botany 91 : 1599 – 1613 . nonaquatic family Chloranthaceae, the infl orescences are en- Buzgo , M. , A. S. Chanderbali , S. Kim , Z. Zheng , D. G. Oppenheimer , closed in early development by the fused stipules of adjacent P. S. Soltis , and D. E. Soltis . 2007 . Floral developmental mor- leaf pairs, which form a sheathing structure. In many species of phology of Persea americana (avocado, Lauraceae): The oddities Hedyosmum, the unistaminate male fl owers, which lack a sub- of male organ identity. International Journal of Plant Sciences 168 : tending bract, are protected by a massive sterile apical part ( En- 261 – 284 . Buzgo , M. , and P. K. Endress . 2000 . Floral structure and development of dress, 1987b , 2008b ). Acoraceae and its systematic relationships with basal angiosperms. Concerning vegetative parts, submerged leaves of water International Journal of Plant Sciences 161 : 23 – 41 . plants tend to be either entire and linear or dissected with linear Buzgo , M. , P. S. Soltis , and D. E. Soltis . 2004 . Floral developmental lobes; thus the presence of linear parts is characteristic. Both morphology of Amborella trichopoda (Amborellaceae). International modes occur, e.g., in aquatic species of Ranunculus (R. reptans Journal of Plant Sciences 165 : 925 – 947 . entire and linear, R. fl uitans dissected with linear lobes), and Caddick , L. R. , P. J. Rudall , P. Wilkin , T. A. J. Hedderson , and M. W. also in Nymphaeales (Hydatellaceae entire and linear, Cabomba Chase . 2002a . Phylogenetics of Dioscoreales based on combined dissected with linear lobes, in addition to peltate, fl oating analyses of morphological and molecular data. Botanical Journal of leaves). Archaefructus is also dissected with linear lobes. the Linnean Society 138 : 123 – 144 . These observations, together with the evidence from our Canright , J. E. 1952 . The comparative morphology and relationships of the Magnoliaceae. I. Trends of specialization in the stamens. American phylogenetic analysis that Archaefructus may be related to Hy- Journal of Botany 39 : 484 – 497 . datellaceae or Ceratophyllum, strengthen the view that its sim- Cantino , P. D. , J. A. Doyle , S. W. Graham , W. S. Judd , R. G. ple fl owers are the result of reduction in an aquatic habitat. Olmstead , D. E. Soltis , P. S. Soltis , and M. J. Donoghue . However, the correlations among fl oral features and the fact 2007 . Towards a phylogenetic nomenclature of Tracheophyta. that our results were sensitive to assumptions concerning the Taxon 56 : 822 – 846 . January 2009] Endress and Doyle — Ancestral flowers 51

Chanderbali , A. S. , S. Kim , M. Buzgo , Z. Zheng , D. G. Oppenheimer , D. E. Daumann , E. 1970 . Das Bl ü tennektarium der Monocotyledonen unter Soltis , and P. S. Soltis . 2006 . Genetic footprints of stamen ancestors besonderer Berü cksichtigung seiner systematischen und phyloge- guide perianth evolution in Persea (Lauraceae). International Journal netischen Bedeutung. Feddes Repertorium 80 : 463 – 590 . of Plant Sciences 167 : 1075 – 1089 . Degtjareva , G. V. , T. H. Samigullin , D. D. Sokoloff , and C. M. Chase , M. W. , M. F. Fay , D. S. Devey , O. Maurin , N. Ronsted , T. J. Valiejo-Roman. 2004 . Gene sampling versus taxon sampling: Is Davies , Y. Pillon, et al. 2006 . Multigene analyses of monocot rela- Amborella (Amborellaceae) a sister group to all other extant angio- tionships: A summary. Aliso 22 : 63 – 75 . sperms? Botanicheskii Zhurnal 89 : 896 – 907 . Chase , M. W. , D. E. Soltis , R. G. Olmstead , D. Morgan , D. H. Les , Donoghue , M. J. , and J. A. Doyle . 1989 . Phylogenetic analysis of an- B. D. Mishler , M. R. Duvall et al. 1993 . Phylogenetics of seed giosperms and the relationships of Hamamelidae. In P. R. Crane and plants: An analysis of nucleotide sequences from the plastid gene S. Blackmore [eds.], Evolution, systematics, and fossil history of the rbcL. Annals of the Missouri Botanical Garden 80 : 528 – 580 . Hamamelidae, vol. 1, 17 – 45. Clarendon Press, Oxford, UK. Chassat , J.-F. 1962 . Recherches sur la ramifi cation chez les Nymphaeacé es. Donoghue , M. J. , and J. A. Doyle . 2000 . Seed plant phylogeny: Demise M é moires de la Soci é t é Botanique de France 1962 : 72 – 95 . of the anthophyte hypothesis? Current Biology 10 : R106 – R109 . Chen , J.-M. , G. W. Robert , and Q.-F. Wang. 2004 . Evolution of aquatic Doty , M. S. , and B. C. Stone . 1966 . Two new species of Halophila life-forms in Alismatidae: Phylogenetic estimation from chloroplast rbcL (Hydrocharitaceae). Brittonia 18 : 303 – 306 . gene sequence data. Israel Journal of Plant Sciences 52 : 323 – 329 . Douglas , A. W. 1995 . Morphological features [Proteaceae]. In P. McCarthy Chen , L. , Y. Ren , P. K. Endress , X. H. Tian , and X. H. Zhang . [ed.], Flora of Australia, vol. 16, 14 – 20. CSIRO, Melbourne, Australia. 2007 . Floral development of Tetracentron sinense (Trochodendraceae) Doyle , J. A. 1978 . Origin of angiosperms. Annual Review of Ecology and and its systematic signifi cance. Plant Systematics and Evolution 264 : Systematics 9 : 365 – 392 . 183 – 193 . Doyle , J. A. 1992 . Revised palynological correlations of the lower Coen , E. , and E. M. Meyerowitz . 1991 . The war of the whorls: Genetic Potomac Group (USA) and the Cocobeach sequence of Gabon interactions controlling fl ower development. Nature 353 : 31 – 37 . (Barremian-Aptian). Cretaceous Research 13 : 337 – 349 . Coiffard , C. , B. Gomez , and F. Thevenard . 2007 . Early Cretaceous Doyle , J. A. 1994 . Origin of the angiosperm fl ower: A phylogenetic per- angiosperm invasion of western Europe and major environmental spective. Plant Systematics and Evolution ( Supplement ) 8: 7 – 29 . changes. Annals of Botany 100 : 545 – 553 . Doyle , J. A. 1996 . Seed plant phylogeny and the relationships of Gnetales. Cook , C. D. K. 1979 . A revision of the genus Rotala (Lythraceae). International Journal of Plant Sciences 157 ( Supplement ): S3 – S39 . Boissiera 29 : 1 – 156 . Doyle , J. A. 2001 . Signifi cance of molecular phylogenetic analyses Cook , C. D. K. 1988 . Wind pollination in aquatic angiosperms. Annals of for paleobotanical investigations on the origin of angiosperms. the Missouri Botanical Garden 75 : 768 – 777 . Palaeobotanist 50 : 167 – 188 . Cook , C. D. K. 1990 . Aquatic plant book. SPB Academic Publishing, The Doyle , J. A. 2005 . Early evolution of angiosperm pollen as inferred Hague, Netherlands. from molecular and morphological phylogenetic analyses. Grana 44 : Cook , C. D. K. 1998a . Butomaceae. In K. Kubitzki [ed.], The families 227 – 251 . and genera of vascular plants, vol. 4, 100– 102. Springer, Berlin, Doyle , J. A. 2006 . Seed ferns and the origin of angiosperms. Journal of Germany. the Torrey Botanical Society 133 : 169 – 209 . Cook , C. D. K. 1998b . Hydrocharitaceae. In K. Kubitzki [ed.], The fami- Doyle , J. A. 2007 . Systematic value and evolution of leaf architecture lies and genera of vascular plants, vol. 4, 234 – 248. Springer, Berlin, across the angiosperms in light of molecular phylogenetic analyses. Germany. Courier Forschungsinstitut Senckenberg 258 : 21 – 37 . Cook , C. D. K. 1999 . The number and kinds of embryo-bearing plants Doyle , J. A. 2008. Integrating molecular phylogenetic and paleobotani- which have become aquatic: A survey. Perspectives in Plant Ecology, cal evidence on origin of the fl ower. International Journal of Plant Evolution and Systematics 2 : 79 – 102 . Sciences 169: 816 – 843. Cook , C. D. K. , and R. Rutishauser . 2007 . Podostemaceae. In K. Kubitzki Doyle , J. A. , P. Bygrave , and A. Le Thomas . 2000 . Implications of mo- [ed.], The families and genera of vascular plants, vol. 9, 304 – 344. lecular data for pollen evolution in Annonaceae. In M. M. Harley, C. Springer, Berlin, Germany. M. Morton, and S. Blackmore [eds.], Pollen and spores: Morphology Crane , P. R. 1985 . Phylogenetic analysis of seed plants and the origin of the and biology, 259 – 284. Royal Botanic Gardens, Kew, UK. angiosperms. Annals of the Missouri Botanical Garden 72 : 716 – 793 . Doyle , J. A. , and M. J. Donoghue . 1986 . Seed plant phylogeny and the Crepet , W. L. , K. C. Nixon , and M. A. Gandolfo . 2004 . Fossil evi- origin of angiosperms: An experimental cladistic approach. Botanical dence and phylogeny: The age of major angiosperm clades based Review 52 : 321 – 431 . on mesofossil and macrofossil evidence from Cretaceous deposits. Doyle , J. A. , H. Eklund , and P. S. Herendeen . 2003 . Floral evolu- American Journal of Botany 91 : 1666 – 1682 . tion in Chloranthaceae: Implications of a morphological phylogenetic Crepet , W. L. , K. C. Nixon , and M. A. Gandolfo . 2005 . An ex- analysis. International Journal of Plant Sciences 164 ( Supplement ): tinct calycanthoid taxon, Jerseyanthus calycanthoides, from the S365 – S382 . Late Cretaceous of New Jersey. American Journal of Botany 92 : Doyle , J. A. , and P. K. Endress . 2000 . Morphological phylogenetic anal- 1475 – 1485 . ysis of basal angiosperms. International Journal of Plant Sciences Crisp , M. D. , and L. G. Cook . 2005 . Do early branching lineages signify 161 ( Supplement ): S121 – S153 . ancestral traits? Trends in Ecology & Evolution 20 : 122 – 128 . Doyle , J. A. , and P. K. Endress . 2007 . Integrating Early Cretaceous fos- Cronquist , A. 1968 . The evolution and classifi cation of fl owering plants. sils into the phylogeny of Recent angiosperms. In Proceedings of Plant Houghton Miffl in, Boston, Massachusetts, USA. Biology & Botany 2007, annual meeting of the Botanical Society of Cutter , E. G. 1957a . Studies of morphogenesis in the Nymphaeaceae. I. America, Chicago, Illinois, USA, abstract no. 150. Website http:// Introduction: Some aspects of the morphology of Nuphar lutea (L.) www.2007.botanyconference.org/engine/search /. Sm. and Nymphaea alba L. Phytomorphology 7 : 45 – 56 . Doyle , J. A. , and L. J. Hickey. 1976 . Pollen and leaves from the Mid- Cutter , E. G. 1957b . Studies of the morphogenesis in the Nymphaeaceae. Cretaceous Potomac Group and their bearing on early angiosperm evo- II. Floral development in Nuphar and Nymphaea: Bracts and calyx. lution. In C. B. Beck [ed.], Origin and early evolution of angiosperms, Phytomorphology 7 : 57 – 73 . 139 – 206. Columbia University Press, New York, New York, USA. Cutter , E. G. 1959 . Studies of morphogenesis in the Nymphaeaceae. IV. Drinnan , A. N. , P. R. Crane , and S. B. Hoot . 1994 . Patterns of fl oral Early fl oral development in species of Nuphar. Phytomorphology 9 : evolution in the early diversifi cation of non-magnoliid dicotyledons 263 – 275 . (eudicots). Plant Systematics and Evolution (Supplement ) 8: 93 – 122 . Cutter , E. G. 1961 . The inception and distribution of fl owers in the Duvall , M. , S. Mathews , N. Mohammad , and T. Russell . Nymphaeaceae. Proceedings of the Linnean Society of London 172 : 2006 . Placing the monocots: Confl icting signal from trigenomic 93 – 100 . analyses. Aliso 22 : 79 – 90 . 52 American Journal of Botany [Vol. 96

Eklund , H. , J. A. Doyle , and P. S. Herendeen . 2004 . Morphological phy- Friedman , W. E. 2008 . Hydatellaceae are water lilies with gymnosper- logenetic analysis of living and fossil Chloranthaceae. International mous tendencies. Nature 453 : 94 – 97 . Journal of Plant Sciences 165 : 107 – 151 . Friedman , W. E. , and J. H. Williams . 2003 . Modularity of the angio- Endress , P. K. 1969 . Gesichtspunkte zur systematischen Stellung der sperm female gametophyte and its bearing on the early evolution of Eupteleaceae (Magnoliales). Untersuchungen über Bau und Entwicklung endosperm in fl owering plants. Evolution 57 : 216 – 230 . der generativen Region bei Euptelea polyandra Sieb. et Zucc. Berichte Friedman , W. E. , and J. H. Williams . 2004 . Developmental evolution of der Schweizerischen Botanischen Gesellschaft 79 : 229 – 278 . the sexual process in ancient fl owering plant lineages. Plant Cell 16 Endress , P. K. 1977 . Ü ber Bl ü tenbau und Verwandtschaft der ( Supplement ): S119 – S132 . Eupomatiaceae und Himantandraceae (Magnoliales). Berichte der Friis , E. M. , and P. R. Crane . 2007 . New home for tiny aquatics. Nature Deutschen Botanischen Gesellschaft 90 : 83 – 103 . 446 : 269 – 270 . Endress , P. K. 1984 . The role of inner staminodes in the fl oral display of Friis , E. M. , P. R. Crane , and K. R. Pedersen . 1991 . Stamen diversity and some relic Magnoliales. Plant Systematics and Evolution 146 : 269 – 282 . in situ pollen of Cretaceous angiosperms. In S. Blackmore and S. H. Endress , P. K. 1986a . Reproductive structures and phylogenetic sig- Barnes [eds.], Pollen and spores: Patterns of diversifi cation, 197 – 224. nifi cance of extant primitive angiosperms. Plant Systematics and Clarendon Press, Oxford, UK. Evolution 152 : 1 – 28 . Friis , E. M. , P. R. Crane , K. R. Pedersen , S. Bengtson , P. C. J. Donoghue , Endress , P. K. 1987a . Floral phyllotaxis and fl oral evolution. Botanische G. W. Grimm , and M. Stampanoni. 2007 . Phase-contrast X-ray Jahrb ü cher f ü r Systematik 108 : 417 – 438 . microtomography links Cretaceous seeds with Gnetales and Endress , P. K. 1987b . The Chloranthaceae: Reproductive structures and Bennettitales. Nature 450 : 549 – 552 . phylogenetic position. Botanische Jahrb ü cher f ü r Systematik 109 : Friis , E. M. , J. A. Doyle , P. K. Endress , and Q. Leng . 2003 . Archaefructus – 153 – 226 . Angiosperm precursor or specialized early angiosperm? Trends in Endress , P. K. 1994a . Diversity and evolutionary biology of tropical fl ow- Plant Science 8 : 369 – 373 . ers. Cambridge University Press, Cambridge, UK. Friis , E. M. , K. R. Pedersen , and P. R. Crane . 1994 . Angiosperm fl oral Endress , P. K. 1994b . Evolutionary aspects of the fl oral structure in structures from the Early Cretaceous of Portugal. Plant Systematics Ceratophyllum. Plant Systematics and Evolution ( Supplement ) 8 : and Evolution ( Supplement ) 8 : 31 – 49 . 175 – 183 . Friis , E. M. , K. R. Pedersen , and P. R. Crane . 2006 . Cretaceous Endress , P. K. 2001 . The fl owers in extant basal angiosperms and infer- angiosperm fl owers: Innovation and evolution in plant reproduc- ences on ancestral fl owers. International Journal of Plant Sciences tion. Palaeogeography, Palaeoclimatology, Palaeoecology 232 : 162 : 1111 – 1140 . 251 – 293 . Endress , P. K. 2003 . Early fl oral development and the nature of the ca- Frohlich , M. W. 2003 . An evolutionary scenario for the origin of fl ow- lyptra in Eupomatiaceae. International Journal of Plant Sciences 164 : ers. Nature Reviews Genetics 4 : 559 – 566 . 489 – 503 . Frohlich , M. W. 2006 . Recent developments regarding the evolutionary Endress , P. K. 2004 . Structure and relationships of basal relictual angio- origin of fl owers. Advances in Botanical Research 44 : 63 – 127 . sperms. Australian Systematic Botany 17 : 343 – 366 . Frohlich , M. W. , and M. W. Chase . 2007 . After a dozen years of prog- Endress , P. K. 2005 . Carpels of Brasenia (Cabombaceae) are com- ress the origin of angiosperms is still a great mystery. Nature 450 : pletely ascidiate despite a long stigmatic crest. Annals of Botany 96 : 1184 – 1189 . 209 – 215 . Frohlich , M. W. , and D. S. Parker. 2000 . The Mostly Male theory of Endress , P. K. 2006 . Angiosperm fl oral evolution: Morphological devel- fl ower evolutionary origins; from genes to fossils. Systematic Botany opmental framework. Advances in Botanical Research 44 : 1 – 61 . 25 : 155 – 170 . Endress , P. K. 2008a. Perianth biology in the basalmost extant angio- Gaussen , H. 1946 . Les Gymnospermes, actuelles et fossiles. Travaux du sperms. International Journal of Plant Sciences 169: 844 – 862. Laboratoire Forestiè re Toulouse, Tome II, É tudes Dendrologiques, Endress , P. K. 2008b . The whole and the parts: Relationships between sec. 1, vol. 1, fasc. 3, chap. 5, 1– 26. Faculté des Sciences, Allé es Saint- fl oral architecture and fl oral organ shape, and their repercussions on Michel, Toulouse, France. the interpretation of fragmentary fl oral fossils. Annals of the Missouri Gomez , B. , V. Daviero-Gomez , C. Martí n-Closas , and M. de la Fuente . Botanical Garden 95 : 101 – 120 . 2006 . Montsechia vidalii, an early aquatic angiosperm from the Endress , P. K. , and J. A. Doyle . 2007 . Floral phyllotaxis in basal an- Barremian of Spain. In Z. Kvač ek, J. Kvač ek, J. Daš kov á , V. Teodoridis, giosperms — Development and evolution. Current Opinion in Plant O. Fatka, J. Bek, S. Opluš til, et al. [eds.], 7th European Palaeobotany- Biology 10 : 52 – 57 . Palynology Conference, Prague, Abstracts, p. 49. National Museum, Endress , P. K. , and A. Igersheim . 2000a . Gynoecium structure and evo- Prague, Czech Republic. lution in basal angiosperms. International Journal of Plant Sciences Goremykin , V. V. , K. I. Hirsch-Ernst , S. Wö lfl , and F. H. Hellwig . 161 ( Supplement ): S211 – S223 . 2003 . Analysis of the Amborella trichopoda chloroplast genome se- Endress , P. K. , and A. Igersheim . 2000b . The reproductive structures of the quence suggests that Amborella is not a basal angiosperm. Molecular basal angiosperm Amborella trichopoda (Amborellaceae). International Biology and Evolution 20 : 1499 – 1505 . Journal of Plant Sciences 161 ( Supplement ): S237 – S248 . Graham , S. W. , and R. G. Olmstead. 2000 . Utility of 17 plastid genes for Endress , P. K. , A. Igersheim , F. B. Sampson , and G. E. Schatz . inferring the phylogeny of the basal angiosperms. American Journal of 2000 . Floral structure of Takhtajania and its systematic position in Botany 87 : 1712 – 1730 . Winteraceae. Annals of the Missouri Botanical Garden 87 : 347 – 365 . Grant , V. 1950 . The protection of the ovules in fl owering plants. Evolution Erbar , C. , and P. Leins . 2003 . Hydrostachyaceae. In K. Kubitzki [ed.], 4 : 179 – 201 . The families and genera of vascular plants, vol. 6, 216– 220. Springer, Guo , Y.-H. , and C. D. K. Cook. 1990 . The fl oral biology of Groenlandia Berlin, Germany. densa (L.) Fourreau (Potamogetonaceae). Aquatic Botany 38 : 283 – 288 . Erbar , C. , and P. Leins . 2004 . Callitrichaceae. In J. W. Kadereit [ed.], Hamann , U. 1975 . Neue Untersuchungen zur Embryologie und Systematik The families and genera of vascular plants, vol. 7, 50– 56. Springer, der Centrolepidaceae. Botanische Jahrbü cher fü r Systematik 9 6 : Berlin, Germany. 154 – 191 . Feild , T. S. , and N. C. Arens . 2007 . The ecophysiology of early angio- Hamann , U. 1976 . Hydatellaceae — A new family of Monocotyledoneae. sperms. Plant, Cell & Environment 30 : 291 – 309 . New Zealand Journal of Botany 14 : 193 – 196 . Feild , T. S. , N. C. Arens , J. A. Doyle , T. E. Dawson , and M. J. Donoghue . Hamann , U. 1998 . Hydatellaceae. In K. Kubitzki [ed.], The families and 2004 . Dark and disturbed: A new image of early angiosperm ecology. genera of vascular plants, vol. 4, 231– 234. Springer, Berlin, Germany. Paleobiology 30 : 82 – 107 . Hayes , V. , E. L. Schneider , and S. Carlquist . 2000 . Floral develop- Friedman , W. E. 2006 . Embryological evidence for developmental labil- ment of Nelumbo nucifera (Nelumbonaceae). International Journal of ity during early angiosperm evolution. Nature 441 : 337 – 340 . Plant Sciences 161 ( Supplement ): S183 – S191 . January 2009] Endress and Doyle — Ancestral flowers 53

Haynes , R. R. , L. B. Holm-Nielsen , and D. H. Les. 1998a . Najadaceae. Kim , S. , J. Koh , M.-J. Yoo , H. Kong , Y. Hu , H. Ma , P. S. Soltis , and In K. Kubitzki [ed.], The families and genera of vascular plants, vol. 4, D. E. Soltis . 2005b . Expression of fl oral MADS-box genes in basal 301– 306. Springer, Berlin, Germany. angiosperms: Implications for the evolution of fl oral regulators. Plant Haynes , R. R. , L. B. Holm-Nielsen , and D. H. Les. 1998b . Ruppiaceae. In Journal 43 : 724 – 744 . K. Kubitzki [ed.], The families and genera of vascular plants, vol. 4, Kim , S. , D. E. Soltis , P. S. Soltis , M. J. Zanis , and Y. Suh . 445– 448. Springer, Berlin, Germany. 2004a . Phylogenetic relationships among early-diverging eudicots Haynes , R. R. , D. H. Les , and L. B. Holm-Nielsen. 1998c . Alismataceae. based on four genes: Were the eudicots ancestrally woody? Molecular In K. Kubitzki [ed.], The families and genera of vascular plants, vol. 4, Phylogenetics and Evolution 31 : 16 – 30 . 11– 18. Springer, Berlin, Germany. Kong , H.-Z. , A.-M. Lu , and P. K. Endress . 2002 . Floral organogenesis Haynes , R. R. , D. H. Les , and L. B. Holm-Nielsen . 1998d . Limnocharitaceae. of Chloranthus sessilifolius (Chloranthaceae): With special emphasis In K. Kubitzki [ed.], The families and genera of vascular plants, vol. 4, on the morphological nature of the androecium of Chloranthus. Plant 271– 275. Springer, Berlin, Germany. Systematics and Evolution 232 : 181 – 188 . Haynes , R. R. , D. H. Les , and L. B. Holm-Nielsen . 1998e . Potamogetonaceae. Kramer , E. M. , and E. A. Zimmer . 2006 . Gene duplication and fl oral In K. Kubitzki [ed.], The families and genera of vascular plants, vol. 4, developmental genetics of basal eudicots. Advances in Botanical 409– 415. Springer, Berlin, Germany. Research 44 : 353 – 384 . Haynes , R. R. , D. H. Les , and L. B. Holm-Nielsen. 1998f. Scheuchzeriaceae. Kuo , J. , and A. J. McComb . 1998a . Cymodoceaceae. In K. Kubitzki [ed.], In K. Kubitzki [ed.], The families and genera of vascular plants, vol. 4, The families and genera of vascular plants, vol. 4, 133– 140. Springer, 449– 451. Springer, Berlin, Germany. Berlin, Germany. Haynes , R. R. , D. H. Les , and L. B. Holm-Nielsen . 1998g . Zannichelliaceae. Kuo , J. , and A. J. McComb . 1998b . Posidoniaceae. In K. Kubitzki [ed.], In K. Kubitzki [ed.], The families and genera of vascular plants, vol. 4, The families and genera of vascular plants, vol. 4, 404– 408. Springer, 470– 474. Springer, Berlin, Germany. Berlin, Germany. Hickey , L. J. , and D. W. Taylor. 1996 . Origin of the angiosperm fl ower. In Kuo , J. , and A. J. McComb . 1998c . Zosteraceae. In K. Kubitzki [ed.], D. W. Taylor and L. J. Hickey [eds.], Flowering plant origin, evolution and The families and genera of vascular plants, vol. 4, 496– 502. Springer, phylogeny, 176– 231. Chapman & Hall, New York, New York, USA. Berlin, Germany. Hiepko , P. 1965 . Vergleichend-morphologische und entwicklungsge- Leebens-Mack , J. , L. A. Raubeson , L. Cui , J. V. Kuehl , M. H. Fourcade , schichtliche Untersuchungen ü ber das Perianth bei den Polycarpicae. T. W. Chumley , J. L. Boore, et al. 2005 . Identifying the basal Botanische Jahrb ücher fü r Systematik 84 : 359 – 426 . angiosperm node in chloroplast genome phylogenies: Sampling one ’ s Hilton , J. , and R. M. Bateman . 2006 . Pteridosperms are the backbone way out of the Felsenstein Zone. Molecular Biology and Evolution of seed plant evolution. Journal of the Torrey Botanical Society 133 : 22 : 1948 – 1963 . 119 – 168 . Leroy , J.-F. 1983 . The origin of angiosperms: An unrecognized ancestral Hochuli , P. A. , U. Heimhofer , and H. Weissert . 2006 . Timing of early dicotyledon, Hedyosmum (Chloranthales), with a strobiloid fl ower is angiosperm radiation: Recalibrating the classical succession. Journal living today. Taxon 32 : 169 – 175 . of the Geological Society 163 : 587 – 594 . Les , D. H. , M. A. Cleland , and M. Waycott . 1997 . Phylogenetic stud- Hoot , S. B. , S. Magalló n , and P. R. Crane . 1999 . Phylogeny of basal eu- ies in Alismatidae, II. Evolution of marine angiosperms (seagrasses) dicots based on three molecular data sets: atpB, rbcL, and 18S nuclear and hydrophily. Systematic Botany 22 : 443 – 463 . ribosomal DNA sequences. Annals of the Missouri Botanical Garden Les , D. H. , M. L. Moody , and C. L. Soros . 2006 . A reappraisal of phylo- 86 : 1 – 32 . genetic relationships in the monocotyledon family Hydrocharitaceae Hughes , N. F. 1994 . The enigma of angiosperm origins. Cambridge (Alismatidae). Aliso 22 : 211 – 230 . University Press, Cambridge, UK. Li , G.-S. , Z. Meng , H.-Z. Kong , Z.-D. Chen , G. Theissen , and A.-M. Lu . Igersheim , A. , M. Buzgo , and P. K. Endress . 2001 . Gynoecium diversity 2005 . Characterization of candidate class A, B and E fl oral homeotic and systematics in basal monocots. Botanical Journal of the Linnean genes from the perianthless basal angiosperm Chloranthus spicatus Society 136 : 1 – 65 . (Chloranthaceae). Development Genes and Evolution 215 : 437 – 449 . Igersheim , A. , and P. K. Endress . 1997 . Gynoecium diversity and sys- Lö hne , C. , T. Borsch , and J. H. Wiersema . 2007 . Phylogenetic analysis of tematics of the Magnoliales and winteroids. Botanical Journal of the Nymphaeales using fast-evolving and noncoding chloroplast markers. Linnean Society 124 : 213 – 271 . Botanical Journal of the Linnean Society 154 : 141 – 163 . Irish , V. F. 2006 . Duplications, diversifi cation, and comparative genetics Maas , P. J. M. , and L. Y. T. Westra . 1984 . Studies in Annonaceae. II. of angiosperm MADS-box genes. Advances in Botanical Research A monograph of the genus Anaxagorea A. St. Hil. Part I. Botanische 44 : 129 – 161 . Jahrbü cher fü r Systematik 105 : 73 – 134 . Irish , V. F. , and A. Litt . 2005 . Flower development and evolution: Gene Maddison , D. R. , and W. P. Maddison . 2003 . MacClade 4: Analysis of duplication, diversifi cation and redeployment. Current Opinion in phylogeny and character evolution, version 4.06. Sinauer, Sunderland, Genetics & Development 15 : 454 – 460 . Massachusetts, USA. Iwamoto , A. , A. Shimizu , and H. Ohba . 2003 . Floral development and Maddison , W. P. 1993 . Missing data versus versus missing characters in phyllotactic variation in Ceratophyllum demersum (Ceratophyllaceae). phylogenetic analysis. Systematic Biology 42 : 576 – 581 . American Journal of Botany 90 : 1124 – 1130 . Martí n-Closas , C. 2003 . The fossil record and evolution of freshwater Jansen , R. K. , Z. Cai , L. A. Raubeson , H. Daniell , C. W. dePamphilis , J. plants: A review. Geologica Acta 1 : 315 – 338 . Leebens-Mack , K. F. M ü ller, et al. 2007 . Analysis of 81 genes from Mathews , S. 2006 . The positions of Ceratophyllum and Chloranthaceae 64 plastid genomes resolves relationships in angiosperms and identi- inferred from phytochrome data. In Proceedings of Botany 2006: fi es genome-scale evolutionary patterns. Proceedings of the National Annual meeting of the Botanical Society of America, Chico, Academy of Sciences, USA 104 : 19369 – 19374 . California, USA, p. 238. Website http://www.2006.botanyconference. Ji , Q. , H. Li , L. M. Bowe , Y. Liu , and D. W. Taylor . 2004 . Early Cretaceous org/engine/search /. Archaefructus eofl ora sp. nov. with bisexual fl owers from Beipiao, west- Mathews , S. , and M. J. Donoghue . 1999 . The root of angiosperm ern Liaoning, China. Acta Geologica Sinica 78 : 883 – 896 . phylogeny inferred from duplicate phytochrome genes. Science 286 : Kaul , R. B. 1968 . Floral morphology and phylogeny in the 947 – 950 . Hydrocharitaceae. Phytomorphology 18 : 13 – 35 . Mayo , S. J. , J. Bogner , and P. C. Boyce . 1998 . Araceae. In K. Kubitzki Kim , S. , J. Koh , H. Ma , Y. Hu , P. K. Endress , M. Buzgo , B. A. Hauser, [ed.], The families and genera of vascular plants, vol. 4, 26– 74. et al. 2005a . Sequence and expression studies of A-, B-, and E-class Springer, Berlin, Germany. MADS-box genes in Eupomatia (Eupomatiaceae): Support for Mohr , B. A. R. , and E. M. Friis . 2000 . Early angiosperms from the Lower the bracteate origin of the calyptra. International Journal of Plant Cretaceous Crato Formation (Brazil), a preliminary report. International Sciences 166 : 185 – 198 . Journal of Plant Sciences 161 ( Supplement ): S155 – S167 . 54 American Journal of Botany [Vol. 96

Moore , M. J. , C. D. Bell , P. S. Soltis , and D. E. Soltis . 2007 . Using Schneider , E. L. , S. C. Tucker , and P. S. Williamson . 2003 . Floral devel- plastid genome-scale data to resolve enigmatic relationships among opment in the Nymphaeales. International Journal of Plant Sciences basal angiosperms. Proceedings of the National Academy of Sciences, 164 ( Supplement ): S279 – S292 . USA 104 : 19363 – 19368 . Sculthorpe , C. D. 1967 . The biology of aquatic vascular plants. Edward Nixon , K. C. , W. L. Crepet , D. W. Stevenson , and E. M. Friis . 1994 . A re- Arnold, London, UK. evaluation of seed plant phylogeny. Annals of the Missouri Botanical Soltis , D. E. , H. Ma , M. W. Frohlich , P. S. Soltis , V. A. Albert , D. Garden 81 : 484 – 533 . G. Oppenheimer , N. S. Altman, et al. 2007 . The fl oral genome: An Parkinson , C. L. , K. L. Adams , and J. D. Palmer . 1999 . Multigene anal- evolutionary history of gene duplication and shifting patterns of gene yses identify the three earliest lineages of extant fl owering plants. expression. Trends in Plant Science 12 : 358 – 367 . Current Biology 9 : 1485 – 1488 . Soltis , D. E. , A. Senters , M. Zanis , S. Kim , J. D. Thompson , P. S. Soltis , Philbrick , C. T. , and D. H. Les . 2000 . Phylogenetic studies in Callitriche : L. P. Ronse De Craene, et al. 2003 . Gunnerales are sister to other Implications for interpretation of ecological, karyological amd polli- core eudicots: Implications for the evolution of pentamery. American nation system evolution. Aquatic Botany 68 : 123 – 141 . Journal of Botany 90 : 461 – 470 . Posluszny , U. , W. A. Charlton , and D. K. Jain . 1986 . Morphology and de- Soltis , D. E. , and P. S. Soltis . 2004 . Amborella not a “ basal angiosperm ” ? velopment of the reproductive shoots of Lilaea scilloides (Poir.) Hauman Not so fast. American Journal of Botany 91 : 997 – 1001 . (Alismatidae). Botanical Journal of the Linnean Society 92 : 323 – 342 . Soltis , D. E. , P. S. Soltis , M. W. Chase , M. E. Mort , D. C. Albach , M. Qiu , Y.-L. , O. Dombrowska , J. Lee , L. Li , B. A. Whitlock , F. Bernasconi- Zanis , V. Savolainen, et al. 2000 . Angiosperm phylogeny inferred Quadroni , J. S. Rest, et al. 2005 . Phylogenetic analyses of basal an- from 18S rDNA, rbcL , and atpB sequences. Botanical Journal of the giosperms based on nine plastid, mitochondrial, and nuclear genes. Linnean Society 133 : 381 – 461 . International Journal of Plant Sciences 166 : 815 – 842 . Soltis , D. E. , P. S. Soltis , P. K. Endress , and M. W. Chase . Qiu , Y.-L. , J. Lee , F. Bernasconi-Quadroni , D. E. Soltis , P. S. Soltis , 2005 . Phylogeny and evolution of angiosperms. Sinauer, Sunderland, M. Zanis , E. A. Zimmer, Z. Chen, V. Savolainen, and M. W. Massachusetts, USA. Chase. 1999 . The earliest angiosperms: Evidence from mitochon- Soltis , P. S. , D. E. Soltis , and M. W. Chase . 1999 . Angiosperm phy- drial, plastid and nuclear genomes. Nature 402 : 404 – 407 . logeny inferred from multiple genes as a tool for comparative biology. Qiu , Y.-L. , L. Li , T. A. Hendry , R. Li , D. W. Taylor , M. J. Issa , A. J. Nature 402 : 402 – 404 . Ronen, M. L. Vekaria, and A. M. White. 2006 . Reconstructing Soltis , P. S. , D. E. Soltis , S. Kim , A. Chanderbali , and M. Buzgo . the basal angiosperm phylogeny: Evaluating information content of 2006 . Expression of fl oral regulators in basal angiosperms and the or- mitochondrial genes. Taxon 55 : 837 – 856 . igin and evolution of ABC-function. Advances in Botanical Research Ren , Y. , H.-F. Li , L. Zhao , and P. K. Endress . 2007 . Floral morpho- 44 : 483 – 506 . genesis in Euptelea (Eupteleaceae, Ranunculales). Annals of Botany Staedler , Y. M. , P. H. Weston , and P. K. Endress . 2007 . Floral 100 : 185 – 193 . phyllotaxis and fl oral architecture in Calycanthaceae (Laurales). Renner , S. S. 1999 . Circumscription and phylogeny of the Laurales: International Journal of Plant Sciences 168 : 285 – 306 . Evidence from molecular and morphological data. American Journal Stebbins , G. L. 1974 . Flowering plants: Evolution above the species of Botany 86 : 1301 – 1315 . level. Harvard University Press, Cambridge, Massachusetts, USA. Rohwer , J. G. , and B. Rudolph . 2005 . Jumping genera: The phyloge- Stefanovic , S. , D. W. Rice , and J. D. Palmer . 2004 . Long branch at- netic positions of Cassytha and Neocinnamomum (Lauraceae) based traction, taxon sampling, and the earliest angiosperms: Amborella or on different analyses of trnK intron sequences. Annals of the Missouri monocots? BMC Evolutionary Biology 4 : 35, doi:10.1186/1471-2148- Botanical Garden 92 : 153 – 178 . 4-35 [online] . Ronse De Craene , L. P. 2008 . Homology and evolution of petals in the Sun , G. , D. L. Dilcher , S. Zheng , and Z. Zhou . 1998 . In search of the fi rst core eudicots. Systematic Botany 33 : 301 – 325 . fl ower: A Jurassic angiosperm, Archaefructus , from northeast China. Ronse De Craene , L. P. , D. E. Soltis , and P. S. Soltis . 2003 . Evolution Science 282 : 1692 – 1695 . of fl oral structures in basal angiosperms. International Journal of Sun , G. , Q. Ji , D. L. Dilcher , S. Zheng , K. C. Nixon , and X. Wang . Plant Sciences 164 ( Supplement ): S329 – S363 . 2002 . Archaefructaceae, a new basal angiosperm family. Science Ronse De Craene , L. P. , and L. Wanntorp . 2008 . Morphology and anat- 296 : 899 – 904 . omy of the fl ower of Meliosma (Sabiaceae): Implications for pollina- Sun , G. , S. Zheng , D. L. Dilcher , Y. Wang , and S. Mei . 2001 . Early tion biology. Plant Systematics and Evolution 271 : 79 – 91 . angiosperms and their associated plants from western Liaoning, Rudall , P. J. , M. V. Remizowa , A. S. Beer , E. Bradshaw , D. W. Stevenson , China. Scientifi c and Technological Education Publishing House, T. D. Macfarlane , R. E. Tuckett, et al. 2008 . Comparative ovule Shanghai, China. and megagametophyte development in Hydatellaceae and water lilies Swofford , D. L. 1990 . PAUP: Phylogenetic analysis using parsimony, reveal a mosaic of features among the earliest angiosperms. Annals of version 3.0. Illinois Natural History Survey, Champaign, Illinois, Botany 101 : 941 – 956 . USA. Rudall , P. J. , D. D. Sokoloff , M. V. Remizowa , J. G. Conran , J. I. Davis , Takahashi , M. 1995 . Development of structure-less pollen wall in T. D. Macfarlane , and D. W. Stevenson . 2007 . Morphology of Ceratophyllum demersum L. (Ceratophyllaceae). Journal of Plant Hydatellaceae, an anomalous aquatic family recently recognized as Research 108 : 205 – 208 . an early-divergent angiosperm lineage. American Journal of Botany Takhtajan , A. 1964 . The taxa of the higher plants above the rank of 94 : 1073 – 1092 . order. Taxon 13 : 160 – 164 . Saarela , J. M. , H. S. Rai , J. A. Doyle , P. K. Endress , S. Mathews , A. D. Takhtajan , A. 1969 . Flowering plants: Origin and dispersal. Oliver & Marchant , B. G. Briggs , and S. W. Graham . 2007 . Hydatellaceae Boyd, Edinburgh, UK. identifi ed as a branch near the base of the angiosperm phylogenetic Taylor , D. W. , G. J. Brenner , and S. H. Basha. 2008 . Scutifolium tree. Nature 446 : 312 – 315 . jordanicum gen. et sp. nov. (Cabombaceae), an aquatic fossil plant Saunders , R. M. K. 2000 . Monograph of Schisandra. Systematic Botany from the Lower Cretaceous of Jordan, and the relationships of re- Monographs 58 : 1 – 146 . lated leaf fossils to living genera. American Journal of Botany 9 5 : Sauquet , H. , J. A. Doyle , T. Scharaschkin , T. Borsch , K. W. Hilu , 340 – 352 . L. W. Chatrou , and A. Le Thomas . 2003 . Phylogenetic analy- Taylor , D. W. , and L. J. Hickey . 1992 . Phylogenetic evidence for the sis of Magnoliales and Myristicaceae based on multiple data sets: herbaceous origin of angiosperms. Plant Systematics and Evolution Implications for character evolution. Botanical Journal of the Linnean 180 : 137 – 156 . Society 142 : 125 – 186 . Thanikaimoni , G. 1985 . Palynology and phylogeny. In H. W. E. van Bruggen Scharaschkin , T. , and J. A. Doyle . 2006 . Character evolution in [ed.], Monograph of the genus Aponogeton (Aponogetonaceae), 11 – Anaxagorea (Annonaceae). American Journal of Botany 93 : 36 – 54 . 14. Bibliotheca Botanica 137: 1 – 76. January 2009] Endress and Doyle — Ancestral flowers 55

Theissen , G. , and R. Melzer . 2007 . Molecular mechanisms underlying Wagner , G. P. 2007 . The developmental genetics of homology. Nature origin and diversifi cation of the angiosperm fl ower. Annals of Botany Reviews Genetics 8 : 473 – 479 . 100 : 603 – 619 . Walker , J. W. , and A. G. Walker . 1984 . Ultrastructure of Lower Tobe , H. , T. Jaffr é , and P. H. Raven . 2000 . Embryology of Amborella Cretaceous angiosperm pollen and the origin and early evolution (Amborellaceae): Descriptions and polarity of character states. of fl owering plants. Annals of the Missouri Botanical Garden 71 : Journal of Plant Research 113 : 271 – 280 . 464 – 521 . Tomlinson , P. B. 1969 . On the morphology and anatomy of turtle grass, Wang , H. , and D. L. Dilcher . 2006 . Aquatic angiosperms from the Thalassia testudinum (Hydrocharitaceae): III. Floral morphology and Dakota Formation (Albian, Lower Cretaceous), Hoisington III lo- anatomy. Bulletin of Marine Science 19 : 286 – 305 . cality, Kansas, USA. International Journal of Plant Sciences 167 : Tomlinson , P. B. 1982 . Anatomy of the monocotyledons. VII. Helobiae 385 – 401 . (Alismatidae) (including the seagrasses). Clarendon Press, Oxford, Wanntorp , L. , and L. P. Ronse De Craene . 2005 . The Gunnera fl ower: UK. Key to eudicot diversifi cation or response to pollination mode? Troll , W. 1964 . Die Infl oreszenzen 1. Fischer, Stuttgart, Germany. International Journal of Plant Sciences 166 : 945 – 953 . Upchurch , G. R. Jr ., P. R. Crane , and A. N. Drinnan. 1994 . The megafl ora Weberling , F. 1988 . Infl orescence structure in primitive angiosperms. from the Quantico locality (Upper Albian). Lower Cretaceous Potomac Taxon 37 : 657 – 690 . Group of Virginia. Virginia Museum of Natural History 4 : 1 – 57 . Weberling , F. 1989 . Morphology of fl owers and infl orescences. van Bruggen , H. W. E. 1998 . Aponogetonaceae. In K. Kubitzki [ed.], Cambridge University Press, Cambridge, UK. The families and genera of vascular plants, vol. 4, 21– 25. Springer, Williams , J. H. , and W. E. Friedman . 2004 . The four-celled female Berlin, Germany. gametophyte of Illicium (Illiciaceae; Austrobaileyales): Implications Voesenek , L. A. C. J. , T. D. Colmer , R. Pierik , F. F. Millenaar , and for understanding the origin and early evolution of monocots, eumag- A. J. M. Peeters. 2006 . How plants cope with complete submer- noliids, and eudicots. American Journal of Botany 91 : 332 – 351 . gence. New Phytologist 170 : 213 – 226 . Yamada , T. , H. Tobe , R. Imaichi , and M. Kato . 2001 . Developmental Vogel , S. 1996 . Life in moving fl uids. The physical biology of fl ow, 2nd morphology of the ovules of Amborella trichopoda (Amborellaceae) ed. Princeton University Press, Princeton, New Jersey, USA. and Chloranthus serratus (Chloranthaceae). Botanical Journal of the von Balthazar , M. , and P. K. Endress . 2002a . Development of infl o- Linnean Society 137 : 277 – 290 . rescences and fl owers in Buxaceae and the problem of perianth inter- Zahn , L. M. , H. Kong , J. H. Leebens-Mack , S. Kim , P. S. Soltis , pretation. International Journal of Plant Sciences 163 : 847 – 876 . L. L. Landherr , D. E. Soltis, et al. 2005 . The evolution of the von Balthazar , M. , and P. K. Endress . 2002b . Reproductive struc- SEPALLATA subfamily of MADS-box genes: A preangiosperm origin tures and systematics of Buxaceae. Botanical Journal of the Linnean with multiple duplications throughout angiosperm history. Genetics Society 140 : 193 – 228 . 169 : 2209 – 2223 . von Mering , S. , and J. W. Kadereit . 2006 . The phylogeny of Zanis , M. J. , D. E. Soltis , P. S. Soltis , S. Mathews , and M. J. Juncaginaceae, a cosmopolitan wetland/coastal family. In Proceedings Donoghue . 2002 . The root of the angiosperms revisited. Proceedings of 8th Young Systematists ’ Forum, Natural History Museum, London, of the National Academy of Sciences, USA 99 : 6848 – 6853 . UK, 7 (abstract). Zanis , M. J. , P. S. Soltis , Y.-L. Qiu , E. Zimmer , and D. E. Soltis . Wagner , G. P. 1989 . The biological homology concept. Annual Review of 2003 . Phylogenetic analyses and perianth evolution in basal angio- Ecology and Systematics 20 : 51 – 59 . sperms. Annals of the Missouri Botanical Garden 90 : 129 – 150 .

Appendix 1. Taxa, characters, and sources of data I n the taxon list, we indicate taxa added or subdivided since Doyle and Endress (2000) with an asterisk. We cite here phylogenetic studies on internal relationships that we consulted to estimate ancestral states in characters that vary within the group. In the character list, DE designates character numbers in Doyle and Endress (2000) . When not otherwise indicated, scorings of taxa follow Doyle and Endress (2000) and are based on references cited therein, including most generally Cronquist (1981) and Kubitzki (1993, 1998 ). Sources of data for taxa added or subdivided since Doyle and Endress (2000) are listed either in the taxon list when they are focused on specifi c taxa or under individual characters or groups of characters when they survey characters across many taxa, as most convenient. The data matrix is presented as Table 2 .

Taxa 12 – 15. Chloranthaceae: Endress (1987b), Zhang and Renner (2003), Eklund et al. (2004) . 1. Amborella (= Amborellaceae). 12. Hedyosmum . Subgenus Tafalla (with fused fl oral bracts) assumed to be a derived subgroup. 2. * Cabomba (Cabombaceae). 13. Ascarina . 3. * Brasenia (Cabombaceae). 14. * Sarcandra . 4 – 6. Nymphaeaceae ( Ito, 1987 ; Les et al., 1999 ; Schneider et al., 2003 ). 15. * Chloranthus . 4. Nuphar. 16. * Liriodendron (Magnoliaceae). 5. Barclaya ( Williamson and Schneider, 1994 ). 17. *Magnolioideae (= Magnolia s.l., Magnoliaceae). Rooting uncertain, but 6. Nymphaeoideae (= Nymphaea s. lat.). Ondinea , Euryale , and Victoria analyses agree that Aromadendron , Alcimandra , Manglietia, and Michelia assumed to be nested within Nymphaea ( L ö hne et al., 2007 ). are nested ( Azuma et al., 2001 ; Kim et al., 2001 ). 7. *Hydatellaceae (Hamann, 1975, 1976 ; Rudall et al., 2007, 2008 ; Friedman, 18. Degeneria (= Degeneriaceae). 2008 ). 19. Galbulimima (= Himantandraceae). 8. Austrobaileya (= Austrobaileyaceae). 20. Eupomatia (= Eupomatiaceae). 9. Trimenia (= Trimeniaceae, including Piptocalyx ). 21. Annonaceae. Anaxagorea is assumed to be basal and the ambavioid clade 10. Illicium (= Illiciaceae) ( Oh et al., 2003 ). (including Cananga) sister to the remaining clades (Doyle and Le Thomas, 11. Schisandraceae ( Saunders, 1998 , 2000 ; Liu et al., 2006 ). 1996 ; Doyle et al., 2000 ; Richardson et al., 2004 ). 56 American Journal of Botany [Vol. 96

22. Myristicaceae. Scoring modifi ed based on Sauquet et al. (2003), who 50. Nelumbo (= Nelumbonaceae). showed that the basal split is not between Mauloutchia and rest of the 51. Platanus (= Platanaceae, not including putative Cretaceous relatives). family, as assumed by Doyle and Endress (2000) , but rather between the myristicoid and combined pycnanthoid-mauloutchioid clades. 52. Proteaceae. Scoring based primarily on Bellendena and Persoonioideae, which form either two basal lines or a clade (Hoot and Douglas, 1998; 23. Calycanthoideae (Calycanthaceae). Following APG (2003) and Staedler et Jordan et al., 2005 ). al. (2007) , we include Idiospermum in Calycanthaceae and designate the remaining genera as Calycanthoideae. Chimonanthus assumed to be sister 53. * Tetracentron (Trochodendraceae). to other Calycanthoideae ( Li et al., 2004 ). 54. * Trochodendron (Trochodendraceae). 24. Idiospermum . 55. Buxaceae (not including putative Cretaceous relatives). Buxus (including 25. Atherospermataceae. Daphnandra and Doryphora assumed to be sister to Notobuxus ) assumed to be sister to the remaining taxa, Sarcococca basal the rest of the family, Atherosperma well nested ( Renner et al., 2000 ). in the rest ( von Balthazar et al., 2000 , 2002b). 26. Siparunaceae. Glossocalyx and Siparuna assumed to be sister groups 56. Acorus (= Acoraceae). (Renner, 1998). 57. Tofi eldiaceae ( Zomlefer, 1997c ). 27 – 29. Monimiaceae: internal relationships based on Renner (1998). 58. Butomus (= Butomaceae). 27. Hortonia . 59. * Aponogeton (= Aponogetonaceae) ( van Bruggen, 1998 ). 28. Monimioideae. Peumus assumed to be sister to Monimia and Palmeria 60. * Scheuchzeria (= Scheuchzeriaceae). (Renner, 1998, 2004). 61. Araceae. Based primarily on Gymnostachys , Pothos , Lysichiton , and 29. Mollinedioideae. Hedycarya and Xymalos assumed to be relatively basal Orontium ( French et al., 1995 ). (Renner, 1998, 2004). 62. *Nartheciaceae. Zomlefer (1997b); basal split assumed to be between 30. Gomortega (= Gomortegaceae). Narthecium -Lophiola and Aletris - Metanarthecium (Caddick et al., 31. Lauraceae. Hypodaphis assumed to be basal, next the cryptocaryoid clade 2002a ). ( Rohwer and Rudolph, 2005 ). 63. Dioscoreaceae. Stenomeris , Tacca , and Trichopus plus Dioscorea treated as 32 – 33. Hernandiaceae ( Kubitzki, 1969 ). forming a trichotomy ( Caddick et al., 2002b ). 32. *Hernandioideae (Hernandia and Illigera ). 64. *Melanthiaceae. Schulze (1978), Zomlefer (1997a) ; assumed internal relationships as in Zomlefer et al. (2001) . 33. *Gyrocarpoideae (Gyrocarpus and Sparattanthelium ). 65. *Ceratophyllum (= Ceratophyllaceae). Rutishauser and Sattler (1987) , 34. Winteraceae. Takhtajania assumed to be sister to the rest of the family, Endress (1994b) , Iwamoto et al. (2003) . Tasmannia basal in the rest ( Karol et al., 2000 ). 66. * Archaefructus inf: fertile axis interpreted as an infl orescence. See text for 35. Canellaceae. Rooting uncertain, but Capsicodendron and Cinnamosma references and discussion of scoring. assumed to be well nested ( Karol et al., 2000 ). 67. * Archaefructus fl o: fertile axis interpreted as a (pre)fl ower. 36. Saururaceae. Basal split not between Saururus and the remaining genera but between Saururus - Gymnostachys and Anemopsis - Houttuynia ( Meng 68. * Archaefructus NP: same as Archaefructus inf but with pollen characters et al., 2002 , 2003 ). (59-73) removed. 37. Piperaceae. Zippelia and Manekia (= Sarcorhachis) assumed to be basal, not Zippelia alone ( Jaramillo et al., 2004 ). Characters 38. Lactoris (= Lactoridaceae). 1 (DE 1). Habit (0) tree or shrub, (1) rhizomatous, scandent, or acaulescent. Amborella rescored as (1) based on seedling establishment pattern 39. Asaroideae (Aristolochiaceae). described by Feild et al. (2001) . Berberidaceae rescored as (1) based on 40. Aristolochioideae (Aristolochiaceae). Thottea assumed to be basal ( Neinhuis revised internal relationships. et al., 2005 ). Anatomical characters (2– 4, 6– 8, 14– 15, 21): references in Doyle and Endress 41. Euptelea (= Eupteleaceae). (2000) , especially Metcalfe and Chalk (1950) and Metcalfe (1987) ; Hydatellaceae: Cutler (1969) ; added monocots: Buxbaum (1922 , 1927 ), 42. Papaveraceae (= Papaverales in Doyle and Endress, 2000 ). Pteridophyllum , Tomlinson (1982) ; Ceratophyllum: Ito (1987), Schneider and Carlquist then Hypecoum plus Fumarioideae assumed to be second and fi rst (1996) . outgroups to the remaining Papaveraceae ( Hoot et al., 1997 ). 2 (DE 4). Protoxylem lacunae (0) absent, (1) present. 43. Lardizabalaceae. Sargentodoxa , Decaisnea, and Sinofranchetia in that order assumed to be basal to the remaining genera ( Hoot et al., 1995 ; 3 (DE 14). Pith (0) uniform, (1) septate (plates of sclerenchyma). Chloranthaceae Wang et al., 2002 ). changed from (?) to (0) based on anatomical collections at Harvard and Kew (JAD); Myristicaceae from (0) to (1), Hernandioideae from (?) to (0) 44. * Circaeaster (Circaeasteraceae). Hu and Yang (1987) , Hu et al. (1990) . based on Sauquet et al. (2003) . 45. Menispermaceae. 4 (DE 5). Cambium (0) present, (1) absent. Circaeaster : Foster (1963) . 46. Berberidaceae. Nandina assumed to be linked with Caulophyllum , 5 (DE 16). Sieve tube plastids (0) S-type (starch), (1) PI-type, (2) PII-type. Gymnospermium , and Leontice rather than basal (Kim et al., 2004; Wang Behnke (1981 , 1988 , 1995 , 2000 ). et al., 2007 ). 6 (DE 17 part). Fibers or sclerenchyma in pericyclic area (including modifi ed 47 – 49. Ranunculaceae: Glaucidium and Hydrastis assumed to be sister to the protophloem) of vascular bundles (0) present, (1) absent. rest of the family, within which Xanthorhiza and Coptis are basal (Hoot, 1995 ). 7 (DE 18). Laticifers in stem (0) absent, (1) present. 47. * Glaucidium ( Tamura, 1972 ; Tobe and Keating, 1985 ). 8 (DE 19). Raphide idioblasts (0) absent, (1) present. Prychid and Rudall (1999) . 48. * Hydrastis ( Tobe and Keating, 1985 ). 9 (DE 20 part). Phyllotaxis (0) alternate (spiral or distichous), (1) opposite or 49. *Core Ranunculaceae. whorled. January 2009] Endress and Doyle — Ancestral flowers 57

10 (DE 20 part). Distichous phyllotaxis (0) absent, (1) on some or all branches. cymes (rhipidia), but our observations indicate they have botryoids (1); Characters 9 and 10: spiral in Hydatellaceae confi rmed by Rudall et al. Aristolochioideae: thyrses inferred to be ancestral ( Gonz á lez, 1999 ); (2007); Cabombaceae: Chassat (1962), Richardson (1969), Moseley Myristicaceae: De Wilde (1991); Annonaceae: rhipidia appear to be et al. (1984) , Rutishauser and Sattler (1987) ; monocots: see general ancestral (Doyle and Le Thomas, 1996; Richardson et al., 2004), most references; Circaeaster : spiral based on fi gures in Foster (1968) ; comparable with thyrsoids; Laurales: Endress and Lorence (personal Glaucidium , Hydrastis : Tobe and Keating (1985) ; Proteaceae: changed observations); monocots: Markgraf (1981) , Posluszny (1983) , and from spiral/distichous to spiral based on basal groups (our observations); Remizova and Sokoloff (2003) as well as references listed for taxa; Tetracentron : our observations; Ceratophyllum : Rutishauser and Sattler Circaeaster : thyrsoid ( Tian et al., 2006 ); Nelumbo : special raceme (1987) speculated that the whorled leaves were derived by fragmentation based on Chassat (1962) and Esau and Kosakai (1975); Buxaceae: von of a single leaf, but they and Les (1985) showed that phyllotaxis is initially Balthazar and Endress (2002a). decussate in the seedling. 22 (DE 37 part). Infl orescence (0) solitary fl ower (or occasionally with 1 – 2 11 (DE 22 modifi ed). First appendage(s) on vegetative branch (0) paired lateral lateral fl owers), (1) botryoid, panicle, or thyrsoid (monotelic), (2) raceme, prophylls, (1) single distinct prophyll (adaxial, oblique, or lateral). State spike, or thyrse (polytelic). labeling inadvertently reversed in Doyle and Endress (2000) due to an editing error. Changes in scoring: Cabomba from (?) to (0), Nuphar and 23 (new). Infl orescence partial units (0) single fl owers, (1) cymes. Nymphaeoideae from (0) to (1) (Chassat, 1962); Degeneria from (?) to 24 (new). Pedicel (0) present in some or all fl owers, (1) absent or highly reduced (1), Siparunaceae from (?) to (0) (Sauquet et al., 2003); Trimenia and (fl ower sessile or subsessile). Saururaceae: Tucker et al. (1993) scored Ascarina from (?) to (0) (Eklund et al., 2004). Platanus, mistakenly scored Saururus and Gymnotheca as pedicellate, but Gymnotheca ( Liang, 1994 ) (0) by Doyle and Endress (2000) although we cited Henry (1847) for one is subsessile as defi ned here; Piperaceae: Zippelia has a short pedicel large lateral prophyll, is rescored as (1). Hydatellaceae: based on lateral ( Liang and Tucker, 1995 ), but Manekia is sessile ( Steyermark, 1971 ), prophylls in the reproductive units ( Rudall et al., 2007 ). Ceratophyllum : and pedicellate species of Piper are deeply nested (Jaramillo and Manos, Rutishauser and Sattler (1987) . 2001 ); Buxaceae: von Balthazar and Endress (2002a, b). 12 (new). Leaf base (0) nonsheathing, (1) sheathing (half or more of stem 25 (new). Floral subtending bracts (0) present, (1) present in female, absent in circumference). General references on taxa and our observations. male fl owers, (2) absent in all fl owers. 13 (DE 23 modifi ed). Stipules (0) absent, (1) adaxial/axillary, (2) interpetiolar, For old and new characters of fl oral organization (26 – 47), we consulted references (3) paired cap. State (3), in Magnoliaceae, previously omitted as in Doyle and Endress (2000) and Ronse De Craene et al. (2003) . autapomorphic and uninformative, is added because Magnoliaceae have been split into two taxa. Acorus and Tofi eldiaceae changed from (1) to (0): 26 (DE 38 modifi ed). Sex of fl owers (0) bisexual, (1) unisexual. State (1) covers the “ stipules ” are not more developed than fl anges of the leaf sheath in both structural and functional unisexuality. Taxa scored as having the other monocots (cf. Bharathan, 1996 ). former bisexual and unisexual state are rescored as (0/1), since this state was found only in Trimenia and Lactoris and dissimilar in these, or mixed 14 (DE 24). Axillary squamules (0) absent, (1) present. Hydatellaceae: long with unisexual in Lardizabalaceae. Winteraceae changed from (0/1) to (0) trichomes near the axils but not scales ( Rudall et al., 2007 ). based on the basal position of Takhtajania. Lardizabalaceae changed to 15 (DE 25). Leaf blade (0) bifacial, (1) unifacial. Hydatellaceae scored as unisexual based on relationships assumed here and the fi nding that pollen bifacial based on the triangular shape of primordia (Rudall et al., 2007). in apparent bisexual fl owers of Decaisnea and Sinofranchetia is abortive Added monocots: Zomlefer (1997b) , Rudall and Buzgo (2002) . ( Qin, 1997 ). Leaf architectural characters (16 – 20): general references, Doyle (2007) , and 27 (DE 39 modifi ed). Floral base (0) hypanthium absent, superior ovary, (1) our observations. Hydatellaceae and Ceratophyllum scored as unknown hypanthium present, superior ovary, (2) partially or completely inferior for some characters because of extreme simplicity and incomparability ovary. Saururaceae: changed from (0) to (2) based on evidence Saururus with ordinary leaves. is nested; Melanthiaceae: relationships and data of Zomlefer et al. (2001) imply superior is ancestral; Trochodendraceae: changed from (0/2) to (2) 16 (DE 26). Leaf shape (0) obovate to elliptical to oblong, (1) ovate, (2) linear. because both tepals and stamens are fused to the ovary in Tetracentron , Trimenia changed from (0) to (0/1) because species formerly placed in and although Trochodendron lacks a perianth, its stamens show similar Piptocalyx are ovate ( Eklund et al., 2004 ); Euptelea changed from (0) to fusion ( Endress, 1986b ). (0/1) following Doyle (2007) . 28 (new). Floral receptacle (female portion) (0) short, (1) elongate. Cases of 17 (DE 27 modifi ed). Major venation (0) pinnate with secondaries at more elongate receptacle in Annonaceae appear to be derived ( Doyle and Le or less constant angle, (1) palmate (actinodromous or acrodromous) or Thomas, 1996 ). crowded (pinnate with crowded basal secondaries, upward decreasing angle), (2) parallel (lateral veins departing at low angles from the midrib 29 (new). Cortical vascular system (0) absent or supplying perianth only, (1) and converging and fusing apically). Parallel added as discussed in Doyle supplying androecium, (2) supplying androecium plus gynoecium. Data (2007). Buxaceae changed from (0/1) to (1) based on presence of both from Ronse De Craene et al. (2003). Annonaceae scored (0/1) based on states in Buxus and palmate in other genera. absence in Anaxagorea and presence in Cananga ( Deroin, 1991 ). 18 (new). Fine venation (0) reticulate, (1) open dichotomous in some or all 30 (new). Floral apex (0) used up after production of carpels, (1) protruding in leaves. Defi nitions of characters 18– 20 assume that the occurrence of the mature fl ower. Unicarpellate taxa scored as unknown. (1) state in some but not all leaves of an individual is potential evidence 31 (DE 41 part). Perianth (0) present, (1) absent. See text for discussion. for relationship. Eupomatia changed from one cycle (DE 41) to absent, based on evidence 19 (DE 28 modifi ed). Base of blade (0) not peltate, (1) peltate in some or all that the calyptra is a bract ( Endress, 2003 ; Kim et al., 2005a); Galbulimima leaves. from one to unknown, because the two calyptrate outer organs appear to be bracts (Endress, 1977, 2003 ), but the petaloid parts might be either 20 (DE 29 modifi ed). Leaf dissection (0) simple, (1) some or all leaves lobed outer staminodes or tepals; Trochodendron scored as unknown, since it is or compound. unclear whether the small nubs below the stamens ( Endress, 1986b ; Wu et 21 (DE 34). Asterosclerids in mesophyll (0) absent, (1) present. al., 2007 ) are tepals, staminodes, or bracts. Infl orescence characters (22– 25): references in Doyle and Endress (2000), 32 (DE 40). Perianth phyllotaxis (0) spiral, (1) whorled. See Endress and Doyle particularly Weberling (1988); references cited in text for Nymphaeales, (2007). Barclaya: Williamson and Schneider (1994); Atherospermataceae Chloranthaceae, and Ceratophyllum ; and general references on added from (0/1) to spiral, because the whorled genus Dryadodaphne is nested taxa. See text for discussion. When male and female infl orescences (Renner et al., 2000); Siparunaceae and Monimioideae rescored as unknown differ, we base scoring on the type with more complex structure. because perianth is too reduced to interpret; core Ranunculaceae scored as Variation in Schisandraceae: Weberling (1988), Saunders (1998, spiral based on data of Sch ö ffel (1932), Hiepko (1965), Endress (1995), and 2000 ); Lactoris described by Gonz á lez and Rudall (2001) as having Tamura (1995) in the context of the phylogeny of Hoot (1995) . 58 American Journal of Botany [Vol. 96

33 (DE 42 modifi ed). Perianth merism (0) trimerous, (1) dimerous, (2) Canellaceae from irregular/trimerous to polymerous ( Wilson, 1966 ; polymerous. Spiral taxa scored as unknown. See text for discussion. Both Occhioni, 1994); Myristicaceae from irregular to trimerous, which is most Magnoliaceae (formerly irregular/trimerous) rescored as trimerous, since likely to be ancestral in the family if the original phyllotaxis was whorled spiral taxa appear to be nested in Magnolioideae; Degeneria changed (Sauquet, 2003); Annonaceae from irregular to trimerous (references from trimerous to unknown because phyllotaxis is spiral; Hernandiaceae for character 41); Hernandiaceae from irregular to polymerous based on from both irregular to Hernandioideae (0/1/2), Gyrocarpoideae (2) based Kubitzki (1969) ; Trochodendron has several stamens per whorl if it is on variation in Kubitzki (1969) ; Hydrastis : Tamura (1995) ; Platanus whorled ( Endress, 1990 ). from 2,4,5-parted to (0/2) because we have excluded the dimerous fossil Quadriplatanus (Magalló n-Puebla et al., 1997) and have not resolved the 43 (new). Number of stamen whorls (series when phyllotaxis is spiral; includes confl icting observations of Sch ö nland (1883) and Bretzler (1924) . inner staminodes) (0) one, (1) two, (2) more than two. Single stamens scored as unknown to avoid redundancy with character 40. See general 34 (DE 41 modifi ed). Perianth whorls (series when phyllotaxis is spiral) (0) references and those for characters 41, 42, and 44. Illicium : reconstructed one, (1) two, (2) more than two. Includes petals (character 36); taxa with ancestral number 11 – 30 ( Oh et al., 2003 ) could represent either (1) or (2); no perianth scored as unknown. Asaroideae changed from two to (0/1) Saururaceae: one whorl in Houttuynia appears to be derived (Meng et al., because small “ petals ” in Asarum are not clearly equivalent to petals of 2002 , 2003 ); Euptelea: ad- and abaxial arcs of stamens suggest one whorl Saruma and not known to be ancestral; Canellaceae from more than two ( Endress, 1986b ; Ren et al., 2007 ). to (1/2) based on data of Gilg (1925) and Wilson (1966) in the context of the phylogeny of Karol et al. (2000); Siparunaceae from one to (0/1), 44 (new). Stamen positions (0) single, (1) double (at least in outer whorl). Monimioideae from more than two to (1/2), because of uncertain merism; Double positions are recognized with reference to a previous or subsequent Hernandioideae from two to (1/2) to allow interpretation of supposedly whorl; thus taxa with no perianth are scored as unknown. Single stamens tetramerous fl owers as dimerous ( Kubitzki, 1969 ; Endress and Lorence, scored as unknown (cf. character 43). See general references and those 2004 ); Ranunculaceae: see character 32; Platanus from two/one to (1/2): for characters 41– 43. Nymphaeoideae uncertain because of high numbers see character 33. refl ecting doubling in the perianth; Winteraceae: single relative to perianth in Takhtajania (Endress et al., 2000) but double in Tasmannia ( Doust, 35 (DE 43 modifi ed). Tepal differentiation (0) all more or less sepaloid; (1) 2000 ) and Pseudowintera ( Vink, 1970 ), Drimys irregular, thus (0/1); outer sepaloid, inner distinctly petaloid; (2) all distinctly petaloid. Does Canellaceae: presence in Cinnamosma and Capsicodendron ( Wilson, not include petals (36). Single sepaloid cycle scored as (0/1). Several taxa 1966 ) presumably derived; Tofi eldiaceae: double in Tofi eldia tenuifolia scored as uncertain to accommodate uncertain interpretations of whorl but not other species (Leinfellner, 1962; Remizova and Sokoloff, 2003); number. Papaveraceae: double in Pteridophyllum , Fumaria , sometimes Hypecoum 36 (new). Petals (0) absent, (1) present. Petals as defi ned here usually have a ( Murbeck, 1912 ); Ranunculaceae: double in Thalictrum presumably narrow base and only one vascular trace, and although initiated acropetally, derived. they usually lag behind sepals and stamens in development ( Hiepko, 1965 ). 45 (DE 48). Stamen fusion (0) free, (1) connate. Taxa with one stamen rescored as Cabomba scored as unknown because the inner organs are delayed but unknown to avoid artifactual steps in reduction of a synandrous androecium otherwise similar to the outer ( Endress, 2001 ), Brasenia not suffi ciently to one. Ascarina: free based on pluristaminate species; Chloranthus : studied; Lardizabalaceae: lack of petals in Decaisnea and Akebia appears unknown because of uncertain interpretation of the androecium (see text; derived based on phylogenetic relationships; Glaucidium : showy parts are Endress, 1987b; Doyle et al., 2003); Aristolochioideae changed from (1) outermost and have several veins and thus not petals ( Hiepko, 1965 ). to (0/1) because apparent fusion in Thottea may be due to fusion to the 37 (DE 45 modifi ed). Nectaries on inner perianth parts (0) absent, (1) present. androgynophore ( Ding Hou, 1981 ; Leins et al., 1988 ; Endress, 1994c ). Cabomba rescored as present: its nectaries are not small, isolated nectar- 46 (DE 70). Inner staminodes (0) absent, (1) present. Taxa with one stamen or secreting areas like the nectarioles of Chimonanthus and Schisandraceae, one whorl of stamens rescored as unknown, since these conditions already with which we compared them in Doyle and Endress (2000) , but are two preclude presence of inner staminodes. Hernandioideae changed from (0) large areas that secrete nectar through special hairs (Vogel, 1998; Endress, to (1) based on recognition in Hernandia (Endress and Lorence, 2004) and 2008a). similar structures alternating with stamens in Illigera ( Kubitzki, 1969 ); 38 (DE 44 part). Outermost perianth parts (0) free, (1) at least basally fused. Gyrocarpoideae from (0) to (0/1) based on presence in Hernandia but not Amborella , Cabomba : Endress (2008a). Sparattanthelium ( Kubitzki, 1969 ). 39 (DE 44 part). Calyptra derived from last one or two bracteate organs below 47 (new). Glandular food bodies on stamens or staminodes (0) absent, (1) the fl ower (0) absent, (1) present. Split from the previous character because present. Calycanthoideae: present on stamens of Calycanthus and it involves parts of apparently different homologies ( Endress, 1977 , 2003 ; Sinocalycanthus but not in Chimonanthus ( Staedler et al., 2007 ). Kim et al., 2005a). Stamen characters (48 – 55): Endress and Hufford (1989) , Hufford and Endress 40 (new). Stamen number (0) more than one, (1) one. See text for discussion. (1989) , Endress (1994c) and references therein, plus the following for individual taxa: Aponogeton , Nartheciaceae, and Melanthiaceae: Endress 41 (DE 46). Androecium phyllotaxis (0) spiral, (1) whorled. See Endress (1996) ; Scheuchzeria : Cronquist (1981) ; Circaeaster : Junell (1931) , Hu and Doyle (2007) . Irregular state of DE 46 eliminated: Annonaceae et al. (1990) ; Hydrastis : Tamura (1995) . rescored as whorled, based on the outer stamens (Endress, 1987a; Erbar and Leins, 1994 ; Leins and Erbar, 1996 ), and Nelumbo , where stamens 48 (DE 49 modifi ed). Stamen base (0) short (2/3 or less the length of anther), arise chaotically on a ring primordium (Hayes et al., 2000), as unknown. (1) long ( > 2/3 length of anther) and wide ( > 1/2 width of anther), (2) long Barclaya : Williamson and Schneider (1994) ; Myristicaceae scored (0/1) (2/3 or more length of anther) and narrow ( < 1/2 width of anther) (typical based not on spiral in Mauloutchia, which now appears derived (Sauquet fi lament). Most scoring changes due to redefi nition of states. Barclaya : et al., 2003), but on possible spiral arrangement in early development base less than half as long as the anther ( Tamura, 1982 ); Austrobaileya : of Myristica (Armstrong and Tucker, 1986); Atherospermataceae: see base of the outer stamens almost as long as the anther; Lactoris : nearly character 32; Siparunaceae rescored as unknown because although sessile ( Bernardello et al., 1999 ); Aristolochioideae changed from Siparuna thecaphora (= andina ) is whorled ( Endress, 1980 ), this species unknown to (0/2) based on variation in Thottea ( Kelly, 1997 ); Eupomatia is nested in the genus, and others are irregular ( Renner et al., 1997 ; rescored (0/1) because of variation between species ( Hiepko, 1965 ; Renner and Hausner, 2005 ); Hydrastis and Glaucidium not suffi ciently Endress, 1994c); Atherospermataceae: all three types represented in studied, possibly chaotic (?); core Ranunculaceae: see character 32; Endress (1994c) , but (2) occurs only in Atherosperma , which is nested; Trochodendron : spiral to approximately whorled ( Endress, 1990 ). Lauraceae: Hypodaphnis , most Beilschmiedia species, and Cryptocarya have a fi lament ( Fouilloy, 1965 ; Hyland, 1989 ); Berberidaceae: (0/2) 42 (DE 47 modifi ed). Androecium merism (0) trimerous, (1) dimerous, (2) because of short base in Nandina ( Hiepko, 1965 ; Terabayashi, 1983 ); polymerous. Spiral taxa scored as unknown. See text for discussion. Dioscoreaceae: (2) because Tacca (short) is too modifi ed to interpret; Nymphaeaceae changed from irregular to polymerous (Endress, Platanus rescored as (0) with exclusion of fi lamentous fossils. 2001); Piperaceae changed from trimerous to (0/1) based on Jaramillo et al. (2004) ; Winteraceae from irregular to polymerous ( Doust, 2001 ); 49 (DE 50). Paired basal stamen glands (0) absent, (1) present. January 2009] Endress and Doyle — Ancestral flowers 59

50 (DE 51 modifi ed). Connective apex (0) extended, (1) truncated or smoothly Sampson (1983) ; Lactoris : Kamelina (1997); Galbulimima : Prakash rounded, (2) peltate. See references for character 48. Nuphar : new peltate et al. (1984); Hortonia: Kimoto and Tobe (2001); Tofi eldiaceae: Wunderlich state; Chloranthaceae: Eklund et al. (2004) ; Magnoliaceae changed from (1936) ; Butomus : Cronquist (1981) ; Aponogeton : van Bruggen (1998) ; (0) to (1) in Liriodendron, based on its relatively truncate apex, (0/1) in Circaeaster : Junell, 1931; Hydrastis : Tobe and Keating (1985) . Magnolioideae based on variation within the group (Endress, 1994c); Pollen characters 59– 73: see Doyle (2005) for updated interpretation and Proteaceae from (0/1) to (1): most basal groups are truncated except references on taxa treated therein. Walker (1976a, b ) and Sampson (2000a) Placospermum, which is presumably derived (Douglas and Tucker, 1996); consulted throughout. Sources of data for taxa added or split since Doyle Platanus rescored as (2) with exclusion of non-peltate fossils. (2005): Hydatellaceae: Linder and Ferguson (1985); Liriodendron and 51 (DE 53). Pollen sacs (0) protruding, (1) embedded. Trimenia and Euptelea , at Magnolioideae: reviewed in Doyle (2005); Aponogeton : Thanikaimoni the limit between states in Doyle and Endress (2000) , have been changed (1985) ; Scheuchzeria : Zavada (1983) , Grayum (1992) ; Nartheciaceae: from unknown to (0) because their sacs are perceptibly more protruding Takahashi and Kawano (1989) , Caddick et al. (1998) ; Dioscoreaceae: Schols than those of otherwise comparable embedded taxa such as Ascarina and et al. (2005); Melanthiaceae: Halbritter and Hesse (1993); Circaeaster : Trochodendraceae (Endress and Sampson, 1983; Endress, 1987b; Hufford Nowicke and Skvarla (1982) ; Glaucidium and Hydrastis : Nowicke and and Endress, 1989 ). Skvarla (1979 , 1981 ); Ceratophyllum : Takahashi (1995) , scored as unknown for structure characters because of extreme exine reduction. 52 (DE 52). Microsporangia (0) four, (1) two. 59 (DE 59). Pollen unit (0) monads, (1) tetrads. 53 (DE 54 modifi ed). Orientation of dehiscence (0) distinctly introrse, (1) latrorse to slightly introrse, (2) extrorse. As discussed in Eklund et al. 60 (DE 62). Pollen size (average) (0) large (> 50 µ m), (1) medium (20– -50 µ m), (2004), including slightly introrse in (1) allows more taxa to be scored (2) small ( < 20 µ m), ordered. Acorus changed from (1) to (2) based on unambiguously, such as Ascarina , Hedyosmum , and Sarcandra , which Erdtman (1952) and Grayum (1992) . would have varied internally under the old defi nition but can now be scored 61 (DE 60 modifi ed). Pollen shape (0) boat-shaped, (1) globose, (2) triangular, as (1). Changes in scoring of Cabombaceae, Nelumbo , Menispermaceae, angulaperturate (Proteaceae). Berberidaceae, and Proteaceae based on reexamination of references in Doyle and Endress (2000) with this new limit between states. This character 62 (DE 61 modifi ed). Aperture type (0) polar (including sulcate, ulcerate, and is diffi cult to score in fl owers that consist of one stamen, because it is disulcate), (1) inaperturate, (2) sulculate, (3) (syn)tricolpate with colpi defi ned relative to the fl oral axis, which is generally not recognizable. The arranged according to Garside’ s law, with or without alternating colpi, more readily visible orientation of the stamen relative to the infl orescence (4) tricolpate. axis may or may not correspond to its orientation in the fl ower, depending 63 (new). Distal aperture shape (0) elongate, (1) round. Taxa with several or no on whether the stamen is located on the abaxial (anterior) or adaxial apertures scored as unknown. (posterior) side of the fl ower. In male fl owers of Hedyosmum and Ascarina (Chloranthaceae), the orientation of the stamen can be inferred 64 (new). Distal aperture branching (0) unbranched, (1) with several branches. from the position of the xylem in the vascular bundle (Endress, 1987b), Taxa with several or no apertures scored as unknown. which implies that the stamens are latrorse to slightly introrse (state 1). 65 (DE 63). Infratectum (0) granular (including “ atectate ” ), (1) intermediate, However, in Ceratophyllum, in which the stamens are extrorse relative to (2) columellar, ordered. the multistaminate male structures, which we interpret as infl orescences, the vascular bundle is too reduced to determine the position of the xylem 66 (DE 64). Tectum (0) continuous or microperforate, (1) perforate (foveolate) to (Endress, 1994b), and we have therefore scored this genus as either semitectate (reticulate), (2) reduced (not distinguishable from underlying introrse or extrorse (0/2). Latrorse stamens (as in Hydatellaceae) can be granules). Glaucidium , Hydrastis , core Ranunculaceae: Nowicke and scored as such without information on stamen orientation. Skvarla (1979 , 1981 ). 54 (DE 55). Mode of dehiscence (0) longitudinal slit, (1) H-valvate, (2) valvate 67 (new). Grading of reticulum (0) uniform, (1) fi ner at ends of sulcus with upward-opening fl aps. Myristicaceae changed from (0/1) to (0) because (liliaceous), (2) fi ner at poles (rouseoid). Scored only in taxa with state (1) H-valvate occurs only in Mauloutchia , now known to be nested (Sauquet in character 66. References cited in Doyle (2005) for taxa covered therein. et al., 2003); Sarcandra and Chloranthus changed from (0) to (1) based Both uniform ( Nandina) and rouseoid ( Leontice , Caulophyllum ) occur on Endress (1987b , 1994c ) and Eklund et al. (2004) ; Liriodendron and in near-basal Berberidaceae ( Nowicke and Skvarla, 1981 ). Scheuchzeria Magnolioideae: Endress (1994c) ; Berberidaceae changed from (0/2) to (2) scored as unknown because it is inaperturate. because the slit dehiscence of Nandina can now be interpreted as derived. 68 (DE 65). Striate muri (0) absent, (1) present. Glaucidium , Hydrastis , core In Calycanthoideae, Sinocalycanthus is H-valvate (Staedler et al., 2007), but Ranunculaceae: Nowicke and Skvarla (1979 , 1981 ). Buxaceae changed this is presumably derived. Ranunculaceae: Tobe and Keating (1985) . from (1) to (0/1) because both states occur in Buxus ( K ö hler, 1981 ; K ö hler 55 (DE 56). Connective hypodermis (0) unspecialized, (1) endothecial or and Br ü ckner, 1982 ). sclerenchymatous. Dioscoreaceae: our observations on Dioscorea . 69 (DE 66). Supratectal spinules (smaller than the width of tectal muri in Characters 56 – 58: Yakovlev (1981 , 1990 ); Amborella : Tobe et al. (2000) ; foveolate-reticulate taxa) (0) absent, (1) present. Cabomba (0/1) based on Siparunaceae: Kimoto and Tobe (2003); Gomortega: Heo et al. (2004); spinules in C. palaeformis ( Ø rgaard, 1991 ); Glaucidium spinules ( Nowicke Hernandioideae: Heo and Tobe (1995) . and Skvarla, 1981 ); Hydrastis smooth ( Nowicke and Skvarla, 1982 ). 56 (DE 57). Tapetum (0) secretory, (1) amoeboid. Furness and Rudall 70 (DE 67). Prominent spines (larger than spinules, easily visible with light (2001) ; Nuphar changed from (1) to (0): Furness and Rudall (2001) ; microscopy) (0) absent, (1) present. Aristolochioideae from (0) to (?): Furness and Rudall (2001) list Asarum 71 (DE 68). Aperture membrane (0) smooth, (1) sculptured. Winteraceae changed as the only member of Aristolochiaceae studied; Atherospermataceae: from (?) to (1) based on the sculptured ring around the ulcus. Winteraceae Furness and Rudall (2001); Hydrastis : Tobe and Keating (1985); changed from (?) to (1) because they have fi ne verrucae either on the ring Ceratophyllum : Shamrov (1983b) . of thickened exine around the pore or across the pore ( Praglowski, 1979 ; Hydrastis 57 (DE 58). Microsporogenesis (0) simultaneous, (1) successive. Nuphar : Sampson, 2000b ); strongly sculptured ( Nowicke and Skvarla, 1982 ), Glaucidium not expanded enough in Nowicke and Skvarla (1981) Batygina and Shamrov (1983); Aponogeton, Nartheciaceae: Furness and to score. Rudall (1999); Scheuchzeria : Yakovlev (1990) ; Melanthiaceae: Eunus (1951) ; Ceratophyllum : Les (1993) . 72 (DE 69 modifi ed). Extra-apertural nexine stratifi cation (0) foot layer, not consistently foliated, no distinctly staining endexine or only problematic 58 (new). Pollen nuclei (0) binucleate, (1) trinucleate. Brewbaker (1967) ; traces, (1) foot layer and distinctly staining endexine, or endexine only, (2) Austrobaileya , Eupomatia , Calycanthoideae, Atherospermataceae, all or in part foliated, not distinctly staining ( Doyle, 2005 ). Monimiaceae, Melanthiaceae: Yakovlev (1981); Cabomba , Brasenia , Nuphar , Barclaya , Nelumbo : Batygina and Shamrov (1983) , Gabarayeva 73 (new). Nexine thickness (0) absent or discontinuous traces, (1) thin (less et al. (2003), Nuphar and Nymphaeoideae scored (0/1) because of confl icts than 1/3 of exine) but continuous, (2) thick (1/3 or more of exine), ordered with Brewbaker (1967) and Yakovlev (1981) ; Trimenia : Endress and ( Doyle, 2005 ). 60 American Journal of Botany [Vol. 96

Old and new gynoecial characters (74– 96): Endress and Igersheim (1997, 1999 , 85 (DE 80). Oil cells in carpels (0) absent or internal, (1) intrusive. Taxa with no 2000a , b ), Igersheim and Endress (1997 , 1998 ), Igersheim et al. (2001) , oil cells in any tissue rescored as unknown. Myristicaceae changed from and references therein, plus the following for individual taxa: Amborella : (0) to (0/1) based on Sauquet et al. (2003) . Endress and Igersheim (2000b), Buzgo et al. (2004); Brasenia : Endress Characters 86 – 88: general gynoecium references cited above and unpublished (2005) ; Winteraceae: Endress et al. (2000) ; Hydatellaceae: Rudall et al. data of Endress and Igersheim. (2007) ; Aponogeton , Scheuchzeria : Igersheim et al. (2001); Melanthiaceae: El-Hamidi (1952); Nartheciaceae: Remizowa et al. (2006) ; Circaeaster , 86 (new). Long unicellular hairs on and/or between carpels (0) absent, (1) Hydrastis : Endress and Igersheim (1999) ; Glaucidium : Tamura (1972) , present. Tobe (2003); Ceratophyllum : Endress (1994b) , Igersheim and Endress 87 (new). Short, curved, appressed, unlignifi ed hairs with up to two short basal (1998) . cells and one long apical cell on carpels (0) absent, (1) present (Endress, 74 (DE 71). Carpel number (0) more than one, (1) one. 2001 ). Endress (2001 , 2005 ); Hydatellaceae: Rudall et al. (2007) . 75 (DE 72). Carpel form (0) ascidiate up to stigma, (1) intermediate (both 88 (new). Nectary on dorsal or lateral sides of carpel or pistillode (0) absent, plicate and ascidiate zones present below the stigma) with ovule(s) on the (1) present. Not found in all Buxaceae, but apparently ancestral (von ascidiate zone, (2) completely plicate, or intermediate with some or all Balthazar and Endress, 2002b). ovule(s) on the plicate zone. 89 (DE 81). Septal nectaries or potentially homologous basal intercarpellary 76 (DE 73 part). Postgenital sealing of carpel (0) none, (1) partial, (2) complete. nectaries (0) absent, (1) present. Nartheciaceae: absent in Lophiola Saarela et al. (2007) scored Hydatellaceae as unknown, but Rudall et al. ( Tamura, 1998 ) and our material of Narthecium , but Utech (1978) reported (2007) confi rmed the lack of postgenital fusion. septal pockets between the carpels in Aletris (including Metanarthecium ), so scored (0/1). 77 (DE 73 part). Secretion in area of carpel sealing (0) present, (1) absent. Rudall et al. (2007) reported no mucilage in Hydatellaceae, but because of 90 (DE 82 modifi ed). Number of ovules per carpel (0) one, (1) two or varying the diffi culty in detecting mucilage and its potential artifactual loss in such between one and two, (2) more than two. This recognizes production of material, we score them as unknown. Trochodendron and Tetracentron : strictly one ovule as most distinctive. Schisandraceae: analyses that nest Endress and Igersheim (1999) . Kadsura in Schisandra ( Liu et al., 2006 ) strengthen the assumption that two ovules are ancestral; Saururaceae: changed from (1/2) to (2) because 78 (DE 74). Pollen tube transmitting tissue (0) not prominently differentiated, (1) the biovulate genus Saururus appears to be nested ( Meng et al., 2002 , one layer prominently differentiated, (2) more than one layer prominently 2003 ); Magnoliaceae were formerly (1), the most parsimonious scoring if differentiated. Liriodendron is (1), Magnolioideae (1/2), but now the two are separated; 79 (DE 75). Style (0) absent (stigma sessile or capitate), (1) present (elongated Araceae changed from (0) to (0/1) because of variation and uncertain apical portion of carpel distinctly constricted relative to the ovary). relationships among basal groups; Euptelea changed from (0/1) to (1) Hedyosmum changed from (0/1) to (1), Asaroideae from (1) to (0/1) because variation between one and rarely two in E. polyandra now falls following Eklund et al. (2004) . In Ceratophyllum Endress (1994b) and in state (1). Iwamoto et al. (2003) showed that the apical extension, on the side where 91 (DE 83 modifi ed). Placentation (0) ventral, (1) laminar-diffuse or “ dorsal. ” the ovule is attached, is ventral and therefore not comparable to a style. “ Dorsal ” ovules, commonly seen in Brasenia ( Endress, 2005 ), are However, the opening of the canal just below this is almost halfway up a attached to the inside of the carpel on its midrib. Ceratophyllum appears long, narrow extension of the carpel above the ovary, which we score as a “ dorsal, ” but development shows it is ventral ( Igersheim and Endress, style (cf. Shamrov, 1983a ). 1998 ; Iwamoto et al., 2003 ). Rudall et al. (2007) reaffi rm that position in 80 (DE 76). Stigma (0) extended (half or more of the style – stigma zone), (1) Hydatellaceae is uncertain. restricted (above slit or around its upper part). Hydatellaceae scored as 92 (DE 84). Ovule direction (0) pendent, (1) horizontal, (2) ascendent. Barclaya unknown because the stigma is reduced to a few long, uniseriate papillae. changed from (1) to (?) based on irregular orientation described by Ceratophyllum scored as unknown because it lacks differentiated stigmatic Igersheim and Endress (1998) . Barclaya changed from (1) to (?) because tissue ( Endress, 1994b ; Iwamoto et al., 2003 ). of excessive variation ( Igersheim and Endress, 1998 ); Dioscoreaceae changed from (0) to (0/1) because Stenomeris appears to be horizontal 81 (DE 77 part). Multicellular stigmatic protuberances or undulations (0) (Dahlgren et al., 1985); Hydrastis has one pendent and one ascendent absent, (1) present. See text for discussion of this and character 82. ovule ( Endress and Igersheim, 1999 ), scored as (0/2). Berberidaceae: Hydrastis -like protuberances in Nandina , Podophyllum , and Jeffersonia but not other genera (Endress and Igersheim, 1999) are 93 (DE 85). Ovule curvature (0) anatropous (or nearly so), (1) orthotropous presumably derived. Hedyosmum and Ascarina have both protuberances (including hemitropous). and unicellular papillae ( Endress, 1987b ). 94 (DE 86). Integuments (0) two, (1) one. 82 (DE 77 part, modifi ed). Stigma papillae (most elaborate type) (0) absent, 95 (DE 91). Chalaza (0) unextended, (1) pachychalazal, (2) perichalazal. (1) unicellular or with a single emergent cell and one or more small Because pachychalazal strictly applies only to anatropous ovules (cf. basal cells, (2) uniseriate pluricellular with emergent portion consisting Periasamy, 1962 ), we have rescored orthotropous taxa as unknown. of two or more cells. State (0) split from unicellular or absent, since lack of papillae is potentially informative with splitting of Sarcandra 96 (DE 92 modifi ed). Nucellus (0) crassinucellar (including weakly so), and Chloranthus and addition of Ceratophyllum; (1) redefi ned to include (1) tenuinucellar or pseudocrassinucellar. Tenuinucellar is defi ned in papillae with small, sunken basal cells, which transfers Cabombaceae terms used for rosids ( Endress and Matthews, 2006 ), with conditions in from pluricellular to unicellular, and Nymphaeoideae from uni/ basal groups corresponding to incompletely tenuinucellar; completely pluricellular to pluricellular. tenuinucellar exists mostly in asterids. Gomortega : Heo et al. (2004) . 83 (DE 78). Extragynoecial compitum (0) absent, (1) present. Annonaceae Fruit and seed anatomy characters (97 – 104) based primarily on Corner (1976) changed from (0/1) to (1) following Sauquet et al. (2003); Buxaceae from and Takhtajan (1985 , 1988 , 1991 ); Hydatellaceae: Hamann (1975) , (?) to (0/1) based on von Balthazar and Endress (2002b); Tofi eldia from Hamann et al. (1979) ; Atherospermataceae, Gomortega : Doweld (2001) ; (0) to (?) based on Igersheim et al. (2001) . Confi rmed in Schisandraceae Tofi eldiaceae: Oganezova (1984); Proteaceae: rescored based on Bellendena ( Lyew et al., 2007 ) and all four genera of Calycanthaceae, including and Persoonioideae as described by Venkata Rao (1960 , 1961 , 1971 ). Idiospermum (Staedler et al., 2009). 97 (DE 93 part). Fruit wall (0) wholly or partly fl eshy, (1) dry. State (1) 84 (DE 79). Carpel fusion (0) apocarpous (including pseudosyncarpous), includes green but not juicy, as in Cabombaceae. Drupes, previously (1) parasyncarpous, (2) eusyncarpous (at least basally). Taxa with one treated as a third state, are specifi ed by the next character. Hedyosmum carpel rescored as unknown to avoid artifactual steps in reduction of a changed from fl eshy/endocarp to dry because the fl eshy tissue is at syncarpous gynoecium to one carpel. Winteraceae rescored (0/1) because the surface of the inferior ovary and may therefore not be gynoecial of parasyncarpy in Takhtajania ( Endress et al., 2000 ). (Endress, 1987b); Saururaceae from fl eshy/dry to fl eshy following January 2009] Endress and Doyle — Ancestral flowers 61

Takhtajan (1988) ; Dioscoreaceae from fl eshy/dry to dry because Myristicaceae are changed from (0) to (2), and Galbulimima is changed berries occur in most but not all members of Tacca , and other taxa are from from (0) to (?) because of probable but reduced ruminations ( Doweld dry (Kubitzki, 1998). and Shevyryova, 1998). Hernandioideae changed from (?) to (1/2) because the ruminations have been variously described as from the chalaza ( Corner, 98 (DE 93 part). Lignifi ed endocarp (0) absent, (1) present. Taxa with dry fruit 1976 ) and the mesotesta ( Takhtajan, 1988 ). wall scored as unknown. Piperaceae changed from fl eshy/endocarp to (0) because descriptions of “ drupes ” do not describe an actual lignifi ed 103 (DE 101). Operculum (0) absent, (1) present. endocarp ( Takhtajan, 1988 ; Prakash and Kin, 1982 ); Proteaceae scored as (1) based on fl eshy persoonioids. 104 (DE 102). Aril (0) absent, (1) present. Myristicaceae changed from (0/1) to (1): an aril is reconstructed as ancestral by Sauquet et al. (2003) . 99 (DE 94 modifi ed). Fruit dehiscence (0) indehiscent or dehiscing irregularly, 105 (new). Female gametophyte (0) four-nucleate, (1) eight- or nine-nucleate. dorsally only, or laterally, (1) dehiscent ventrally or both ventrally and Tetrasporic types in Piperaceae scored as unknown. Williams and dorsally, (2) horizontally dehiscent with vertical extensions. Defi nitions Friedman (2004); Amborella : Friedman (2006) ; Hydatellaceae: Hamann of states (0) and (1) inadvertently reversed in Doyle and Endress (2000); (1975) ; Melanthiaceae: Yakovlev (1990) ; Nartheciaceae: Zomlefer (2) added for Papaveraceae and some Berberidaceae. Hydatellaceae: (1997b) ; Dioscoreaceae: Huber (1998) ; Circaeaster : Junell (1931) , Hu some species dehisce along the vascular bundles of the single carpel and Yang (1987). (Rudall et al., 2007), but whether this is ancestral or derived within Hydatellaceae, it is not comparable with dehiscence in other taxa, 106 – 110: Yakovlev (1981 , 1990 ), Takhtajan (1985 , 1988 , 1991 ); Amborella : Tobe so we score the family as (0). Saururaceae changed from (0/1) to (0) et al. (2000); Hydatellaceae: Hamann (1975); added monocots: Kubitzki because Saururus , the only indehiscent genus (Takhtajan, 1988), now (1998) , Zomlefer (1997a – c ); Ranunculaceae: Tobe and Keating (1985) . appears to be nested; Aristolochioideae from (1) to (?) because they are septicidal rather than ventrally dehiscent (Huber, 1993); Berberidaceae 106 (DE 103). Endosperm development (0) cellular, (1) nuclear, (2) helobial. from indehiscent to (0/2) because Caulophyllum , Gymnospermium , Amborella , Nuphar , Illicium : Floyd and Friedman (2001); Gomortega : Heo Leontice, and many Epimediineae have horizontal dehiscence (Loconte, et al. (2004); Siparunaceae: Kimoto and Tobe (2003); Hernandioideae: 1993 ); Platanus changed from (0/1) to (0) with elimination of dehiscent changed from (1) to (0) based on Heo and Tobe (1995) ; Circaeaster : fossils. Junell (1931) , Hu and Yang (1987) . 100 (DE 95). Testa (0) slightly or nonmultiplicative, (1) multiplicative. 107 (DE 104). Endosperm in mature seed (0) present, (1) absent. Nikiticheva and Scheuchzeria Myristicaceae changed from (1) to (0) based on Sauquet et al. (2003) . Proskurina (1992) described as having endosperm present as a thin fi lm around the embryo, but this is not clearly different from similar 101 (DE 96). Exotesta (0) unspecialized, (1) palisade or shorter sclerotic tissue in Butomus ( Takhtajan, 1985 ), so we score Scheuchzeria as (?). cells, (2) tabular, (3) longitudinally elongated, more or less lignifi ed 108 (DE 105 modifi ed). Perisperm (0) absent, (1) from nucellar ground tissue, cells. State (3) added for Aponogeton and Scheuchzeria (Takhtajan, (2) from nucellar epidermis. State (2) added for Acorus ( Rudall and 1985). Tofi eldiaceae changed from (2) to (0/2); Proteaceae from (0/1) Furness, 1997 ). to (0). 109 (DE 106). Embryo (0) minute (less than 1/2 length of seed interior), (1) large. 102 (DE 100). Ruminations (0) absent, (1) testal, (2) tegminal and/or chalazal. Following Sauquet et al. (2003) , state (1) is restricted to testal ruminations, 110 (DE 107). Cotyledons (0) two, (1) one. 62 American Journal of Botany [Vol. 96

Table 2. Complete data matrix, including infl orescence and fl oral characters and other characters relevant for placement of Ceratophyllum and Archaefructus . A = 0/1, B = 0/2, C = 0/4, D = 1/2, E = 0/1/2. 1 2 3 4 5 6 7 8 9 0 1 12345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890 Amborella 1?000000010000010000010001100000?20001000?20000100000000100110?020?01011100000001210?010000010?001000000100000 Cabomba 11010110A1000?011111020000000001012?110010010?02010020001000000020?1A010100000100100?010021000001?001010020100 Brasenia 1101?11001?0000110100?00?0000001012?000010210002010010000100000020?0001010000?100100?010011000??1?001010?20100 Nuphar 1101011000100?0110001200000000010211000012210000021001000A00000010?01111100200000112?0000210000000001011000100 Barclaya 1101011000?00?0110001200?020000122100000122?00000010000??00112??20?000???012000002?2?010021?10?00000?010000100 Nymphaeoideae 1101011000101001100012002020010122100000122?0001001000000A0012??10?000?1200200000212?010021000A000001011000100 Hydatellaceae 1101210000000002???0021021?00?1???????01??????0201?0100??002000020?010?01100??0?02???01000?000001?00101000010? Austrobaileya 10000000100000010000000000000000?21000000?20010100000000000110002100001110000110011000000201002000010100??0000 Trimenia A000000010000?0A0000010A0A000?00?20000000?D000020000D000000112??210010111100000012??A0A000000000000010000?0000 Illicium 00000000000000000000100000000100?21000000?D0000101000000000113??210000111011011001101000000200001?1A1000000000 Schisandraceae 1000000000000?0000001B0001010000?21000000?E010010100B000000113??2100001110000100011010000101000000001000000000 Hedyosmum A000?00010?12?000000021111200?0100A??001??????000E10101?00011001210010102100001011??0000000010?01?000000100000 Ascarina 1000000010012?000000020101?00?1???????0A??000?000010101???011000210010102100000011??0000000010?000000000??0000 Sarcandra 1000000010012?000000?20100?00?1???????01??????010110110000011???210000102100000000??1000000010?000010000100000 Chloranthus 10000000100120000000020100?00?1???????0???????0A0000010000011???21000011D100000010??1000000010?000000000100000 Liriodendron 0010A000000130000000000000012001021000100?200000011020?00000000020?000021022001101000000010100001?010000100000 Magnolioideae 0010A0000A013000000010000001200102D000100?2000000A100A0000000000D0?0000210220010010000000D0100001?110000100000 Degeneria 00101000011000000000000000001000?21001000?200110001021000000000000?0000?0122110002??00000201000000110100100000 Galbulimima 001000000110000000000000000110????????100?200110001021001001100000?0000?002?010101100??00001000001000?001?0000 Eupomatia 0000100001100000000000000010001???????100?20111A00100100000112??00?0000210?21101021?000002010000000101001?0000 Annonaceae 0010100001100000000011000000A0010210000010210AA000102110A000000000?00002102101A101100A000D0D0020A0A10100100000 Myristicaceae 00100000010000000000120AB1000?0100A??100A00?1?0?010020001001100021000012D112000101??AA000002001000100201110000 Calycanthoideae 000010001000000A0000000000100000?21000000?2001A0000020000A0112??2100000210221011011?0100010200001?010000101010 Idiospermum 00?0100010000?000000?00000100000?2100?000?2001000000200???0112???0?000???A2210011??001000102000?1?00?000??1010 Atherospermatac 00001000100000000000010000100000?21000000?20010A1A11E20000011000210000A21011001101100100000200001?0000001?0000 Siparunaceae 000010001000000000000210?110?00??AA???00????0?0A01110200100211??10?010?210110011011000000002010001000000100000 Hortonia 0000100010?00?011000010000100000?21000000?D001021100200??00111???0?010??001101110110110000000000010000001?0000 Monimioideae 0000A00010000000000001000110?00??DA00000??2?0?0211000100100111??10?001??001100110110110000000000010000001000?0 Mollinedioideae 0000100010?00?000000010001100001DD000?001DDA0?000100000010AD11??1B?0AA??0000000001100A0000000000010000001?00A0 Gomortega 0000100010?00000000001000020?000?20000000??0010211110200000111??10?010??0012101101??0000000010?001000000?00010 Lauraceae 00001000A000000AA000010000200?010100000010200102111A020A100111??02?001??1112121101??0A00000000100A010000111010 Hernandioideae 000010000000000110A0021000200?01EDB000001210010211110201100011??02?001??1112121101??0100000000A001010D00101010 Gyrocarpoideae 00?0100000?00001100A021000200?01200??A0012A00A0211112201100111?????0?1???112121101??01000000000001010000101010 Winteraceae 0000A00000000000000001000000000112100100122A000101002010001110102100001110221100010A00000201000000001000100000 Canellaceae 0000100000000?0000000A00000000010D1001001210100?0100200000011000210000002021010?01?100000D0100000000100A1?0000 Saururaceae 1000000001111001100002010020001???????00101?0002011010000002100020?000102022101001011000020110?000100000100100 Piperaceae 100000000A111?01100002010000001???????001AA?00020110A0000002100020?0101010?210A?01?1100000?210?000000000?A0100 Lactoris 1000000001111?00100001000A00000100A??00010100000000020000111101010?000001022101001000000020100011?100000100000 Asaroideae 100020000110000110000000002000010AAA010010D100020000200000011A002A000011102211A002020000020100001?100000100000 Aristolochioideae 1000100001100001100002100020000100A??10010A1A00B010020??A00111??20?0A0?11022100?01?20000020100001??1A000100000 Euptelea 000000000000000AA00002000000001???????001?0?0?0200001110000114??21001011101210000100?000010000001?002000100000 Papaveraceae 1000001000?A0?0110010D000000000112A1000011A100020100200A000114??2A?0A011D020001?01?1?000020000001?201000110000 Lardizabalaceae A0000000000000011001020001000001022110001010A00201002000000114??20?00011102101010110?0000B01000000011000100000 Circaeaster 1000??0000?00?0?1100010000000000?0A??0000?000?0201010000000114??20?1001??A00000101?0?000010011011?000000100000 Menispermaceae A000000A000000011000010A0100000102A110001010A0020100A000000214??21000011102100A1AA10?A000102000001000A00110010 Berberidaceae 1000000000?1A?01100101000000A?010211A0001010000B0100E20A000114??21B00011D10001A000???000020D000000BA100A110000 Glaucidium 10??0?0001??0?011001?0000000200111200000??2?00020100?0????0114??210010?1202???011?00???0020100?11?101000?10010 Hydrastis 10000?0001?10?01100100000000000100A??000??2?0001010010?0?00114??2101001120221?111000?100020B00001?101000?10000 Core Ranunculac 1000000000?10?011001010000000000?12110000?20000201001000000114??20?01011102211110100?A000E0?00011?101000110000 Nelumbo 10010?1001111?011010000000000000?2100000??2000020000D000010014??21000011100001000100?000000000101?000000111010 Platanus 0000000001111?0010010D0121000001B10000001EA0000002A01110000114??21000011202211100100???0010010?01?000000110010 Proteaceae 0000000000000000100AA20000000?01110000001110000201000000000124??2A0000?02122111101???000010010?0A10A000011A010 Tetracentron 00000000010110011000?D0100200001110000001110000201101110000114??212100112022111001?2?001020000001?1020001?0000 Trochodendron 000000000000000?A0001100002010????????00A220000201101110000114??21210011202201100102?001020000001?102000100000 Buxaceae 0000?000A000000A1000010A01000001110000001110000200000010000114??210A001?102211100102?00101000000A0A010001000A0 Acorus 100120000111011220000201200000010100000010100002010000001002000020?000011012010101?2A00002?010?100000000100201 Tofi eldiaceae 1001200001?10012200002A00000000101000000101A0002010000?00001000021100000102201110100?000120100001?10A000120011 Butomus 110120000111010220000210000000010110000010110002010020011101000021100000102101110200?000121200001?102000121011 Aponogeton 11012110???1010120000201200000010AB0000010100002010020?1A101A00001001010102101110100?000020200001??03000121011 Scheuchzeria 1101200001?10112200001000000?0010100000010100002000020?111?111??21?000???02101110100?000010D00001?10300012?011 Araceae 10012AA101110A0DD000020120000001A10000001A100001010020?1100100002A?000011AA0010A0E?2?0000A0BAA0000000000120011 Nartheciaceae 100120000A?100A2200002000000?0010120000010100002010000?01?01000021100000102101110102?000A20200001?101000120001 Dioscoreaceae 1001200100?AA00110000D00002000010120000010100002010000000001000020?0000010210111010D?0001D0A00001?A000001100A1 Melanthiaceae 1001200100?1000220000200000000010120?00010100002010020?01001000021100010102101110102?000020200001?101000120001 Ceratophyllum 110101001000000??1010201D1?00?1???????01??????000?10B000A00111???????????100001?00???000000011?01?000000100010 Archaefructus inf 1???????0??00?011101?20021?0?01????????0??0?0??000????????010000?0?000???A????1????0?????200????1???1????????? Archaefructus fl o 1???????0??00?011101?0000001??1????????0??2?0??00?????????010000?0?000???0????1????0?????200????1???1????????? Archaefructus NF 1???????0??00?011101?20021?0?01????????0??0?0??000???????????????????????A????1????0?????200????1???1????????? January 2009] Endress and Doyle — Ancestral flowers 63

APPENDIX LITERATURE CITED Doust , A. N. 2000 . Comparative fl oral ontogeny in Winteraceae. Annals of the Missouri Botanical Garden 87 : 366 – 379 . Note: References that appear only in the Appendix are cited Doust , A. N. 2001 . The developmental basis of fl oral variation in Drimys here. References found in both the Appendix and the main text winteri (Winteraceae). International Journal of Plant Sciences 162 : are listed in the main Literature Cited. 697 – 717 . Doweld , A. B. 2001 . Carpology and phermatology of Gomortega Armstrong , J. E. , and S. C. Tucker . 1986 . Floral development in Myristica (Gomortegaceae): Systematic and evolutionary implications. Acta (Myristicaceae). American Journal of Botany 73 : 1131 – 1143 . Botanica Malacitana 26 : 19 – 37 . Azuma , H. , J. G. Garcí a-Franco , V. Rico-Gray , and L. B. Thien . Doweld , A. B. , and N. A. Shevyryova . 1998 . Carpology, seed anatomy 2001 . Molecular phylogeny of the Magnoliaceae: The biogeography and taxonomic relationships of Galbulimima (Himantandraceae). of tropical and temperate disjunctions. American Journal of Botany Annals of Botany 81 : 337 – 347 . 88 : 2275 – 2285 . Doyle , J. A., and A. Le Thomas . 1996 . Phylogenetic analysis and Batygina , T. B. , and I. I. Shamrov . 1983 . Embryology of the character evolution in Annonaceae. Bulletin du Museum National Nelumbonaceae and Nymphaeaceae: Pollen grain structure (some pe- d’ Histoire Naturelle, Paris , sé r. B, Adansonia 18: 279 – 334. culiar features related to development of the pollen grain and anther El-Hamidi , A. 1952 . Vergleichend-morphologische Untersuchungen am wall). Botanicheskii Zhurnal 68 : 1177 – 1183 . Gynoeceum der Unterfamilien Melanthioideae und Asphodeloideae Behnke , H.-D. 1981 . Siebelement-Plastiden, Phloem-Protein und der Liliaceae. Hug, Zurich, Switzerland. Evolution der Bl ü tenpfl anzen: II. Monokotyledonen. Berichte der Endress , P. K. 1980 . Ontogeny, function and evolution of extreme fl oral Deutschen Botanischen Gesellschaft 94 : 647 – 662 . construction in Monimiaceae. Plant Systematics and Evolution 134 : Behnke , H.-D. 1988 . Sieve-element plastids, phloem protein, and evolu- 79 – 120 . tion of fl owering plants: III. Magnoliidae. Taxon 37 : 699 – 732 . Endress , P. K. 1986b . Floral structure, systematics and phylogeny in Behnke , H.-D. 1995 . Sieve-element plastids, phloem proteins, and the Trochodendrales. Annals of the Missouri Botanical Garden 73 : 297 – 324 . evolution of the Ranunculanae. Plant Systematics and Evolution Endress , P. K. 1990 . Patterns of fl oral construction in ontogeny and phy- ( Supplement ) 9: 25 – 37 . logeny. Biological Journal of the Linnean Society 39 : 153 – 175 . Behnke , H.-D. 2000 . Forms and sizes of sieve-element plastids and evo- Endress , P. K. 1994c . Shapes, sizes and evolutionary trends in stamens of lution of the monocotyledons. In K. L. Wilson and D. A. Morrison Magnoliidae. Botanische Jahrb ü cher f ü r Systematik 115 : 429 – 460 . [eds.], Monocots: Systematics and evolution, 163 – 188. CSIRO, Endress , P. K. 1995 . Floral structure and evolution in Ranunculanae. Melbourne, Australia. Plant Systematics and Evolution (Supplement) 9 : 47 – 61 . Bernardello , G. , G. J. Anderson , S. P. Lopez , M. A. Cleland , T. Endress , P. K. 1996 . Diversity and evolutionary trends in angiosperm F. Stuessy , and T. J. Crawford . 1999 . Reproductive biology of anthers. In W. G. D ’ Arcy and R. C. Keating [eds.], The anther: Lactoris fernandeziana (Lactoridaceae). American Journal of Botany Form function and phylogeny, 92 – 110. Cambridge University Press, 86 : 829 – 840 . Cambridge, UK. Bharathan , G. 1996 . Does the monocot mode of leaf development char- Endress , P. K. , and L. D. Hufford . 1989 . The diversity of stamen struc- acterize all monocots? Aliso 14 : 271 – 279 . tures and dehiscence patterns among Magnoliidae. Botanical Journal Bretzler , E. 1924 . Beitr ä ge zur Kenntnis der Gattung Platanus. of the Linnean Society 100 : 45 – 85 . Botanisches Archiv 7 : 388 – 417 . Endress , P. K. , and A. Igersheim . 1997 . Gynoecium diversity and sys- Brewbaker , J. L. 1967 . The distribution and phylogenetic signifi cance of tematics of the Laurales. Botanical Journal of the Linnean Society binucleate and trinucleate pollen grains in the angiosperms. American 125 : 93 – 168 . Journal of Botany 54 : 1069 – 1083 . Endress , P. K. , and A. Igersheim . 1999 . Gynoecium diversity and Buxbaum , F. 1922 . Vergleichende Anatomie der Melanthioideae. Feddes systematics of the basal eudicots. Botanical Journal of the Linnean Repertorium Beihefte 29 : 1 – 80 . Society 130 : 305 – 393 . Buxbaum , F. 1927 . Nachtr ä ge zur vergleichenden Anatomie der Endress , P. K. , and D. H. Lorence . 2004 . Heterodichogamy of a novel type Melanthioideae. Beihefte zum Botanischen Centralblatt 44 : 255 – 263 . in Hernandia (Hernandiaceae) and its structural basis. International Caddick , L. R. , C. A. Furness , K. L. Stobart , and P. J. Rudall . Journal of Plant Sciences 165 : 753 – 763 . 1998 . Microsporogenesis and pollen morphology in Dioscoreales Endress , P. K. , and M. L. Matthews . 2006 . First steps towards a fl o- and allied taxa. Grana 37 : 321 – 336 . ral structural characterization of the major rosid subclades. Plant Caddick , L. R. , P. Wilkin , P. J. Rudall , T. J. A. Hedderson , and Systematics and Evolution 260 : 223 – 251 . M. W. Chase . 2002b . Yams reclassifi ed: A recircumscription of Endress , P. K. , and F. B. Sampson . 1983 . Floral structure and relation- Dioscoreaceae and Dioscoreales. Taxon 51 : 103 – 114 . ships of the Trimeniaceae (Laurales). Journal of the Arnold Arboretum Corner , E. J. H. 1976 . The seeds of dicotyledons. Cambridge University 64 : 447 – 473 . Press, Cambridge, UK. Erbar , C. , and P. Leins . 1994 . Flowers in Magnoliidae and the origin of Cronquist , A. 1981 . An integrated system of classifi cation of fl owering fl owers in other subclasses of the angiosperms. I. The relationships plants. Columbia University Press, New York, USA. between fl owers of Magnoliidae and Alismatidae. Plant Systematics Cutler , D. A. 1969 . Juncales. In C. R. Metcalfe [ed.], Anatomy of the and Evolution ( Supplement ) 8: 193 – 208 . monocotyledons, vol. 4, 1 – 357. Clarendon Press, Oxford, UK. Erdtman , G. 1952 . Pollen morphology and plant taxonomy: Angiosperms. Dahlgren , R. M. T. , H. T. Clifford , and P. F. Yeo . 1985 . The families Almqvist & Wiksell, Stockholm, Sweden. of the monocotyledons: Structure, evolution and taxonomy. Springer, Esau , K. , and H. Kosakai. 1975 . Leaf arrangement in Nelumbo nucifera: A Berlin, Germany. re-examination of a unique phyllotaxy. Phytomorphology 25 : 100 – 112 . De Wilde , W. J. J. E. 1991 . The genera of Myristicaceae as distinguished Eunus , A. M. 1951 . Contributions to the embryology of the Liliaceae. V. by their infl orescences, and the description of a new genus, Bicuiba. Life history of Amaianthium muscaetoxicum Walt. Phytomorphology Beiträ ge zur Biologie der Pfl anzen 66 : 95 – 125 . 1 : 73 – 79 . Deroin , T. 1991 . Floral vasculature of Magnoliales — Preliminary ap- Feild , T. S. , T. Brodribb , T. Jaffr é , and N. M. Holbrook . 2001 . proach of its role in pollination. Comptes Rendus de l’ Acadé mie des Acclimation of leaf anatomy, photosynthetic light use, and xy- Sciences, Sé rie III 312 : 355 – 360 . lem hydraulics to light in Amborella trichopoda (Amborellaceae). Ding Hou . 1981 . Florae Malesianae Praecursores LXII. On the genus International Journal of Plant Sciences 162 : 999 – 1008 . Thottea (Aristolochiaceae). Blumea 27 : 301 – 332 . Floyd , S. K. , and W. E. Friedman . 2001 . Developmental evolu- Douglas , A. W. , and S. C. Tucker . 1996 . Comparative fl oral ontogenies tion of endosperm in basal angiosperms; evidence from Amborella among Persoonioideae including Bellendena (Proteaceae). American (Amborellaceae), Nuphar (Nymphaeaceae), and Illicium (Illiciaceae). Journal of Botany 83 : 1528 – 1555 . Plant Systematics and Evolution 228 : 153 – 169 . 64 American Journal of Botany [Vol. 96

Foster , A. S. 1963 . The morphology and relationships of Circaeaster. Huber , H. 1998 . Dioscoreaceae. In K. Kubitzki [ed.], The families Journal of the Arnold Arboretum. 44 : 299 – 321 . and genera of vascular plants, vol. 3, 216– 235. Springer, Berlin, Foster , A. S. 1968 . Further morphological studies on anastomoses in Germany. the dichotomous venation of Circaeaster. Journal of the Arnold Hufford , L. D. , and P. K. Endress . 1989 . The diversity of anther struc- Arboretum. 49 : 52 – 67 . tures and dehiscence patterns among Hamamelididae. Botanical Fouilloy , R. 1965 . Laurac é es. In A. Aubr é ville [ed.], Flore du Gabon, Journal of the Linnean Society 99 : 301 – 346 . vol. 10, 7 – 81. Mus é um National d ’ Histoire Naturelle, Paris, France. Hyland , B. P. M. 1989 . A revision of Lauraceae in Australia (excluding French , J. C. , M. G. Chung , and Y. K. Hur . 1995 . Chloroplast DNA phy- Cassytha ). Australian Systematic Botany 2 : 135 – 367 . logeny of the Arifl orae. In P. J. Rudall, P. J. Cribb, D. F. Cutler, and Igersheim , A. , and P. K. Endress . 1998 . Gynoecium diversity and sys- C. J. Humphries [eds.], Monocotyledons: Systematics and evolution, tematics of the paleoherbs. Botanical Journal of the Linnean Society 255 – 275. Royal Botanic Gardens, Kew, UK. 127 : 289 – 370 . Furness , C. A. , and P. J. Rudall . 1999 . Microsporogenesis in mono- Ito , M. 1987 . Phylogenetic systematics of the Nymphaeales. Botanical cotyledons. Annals of Botany 84 : 475 – 499 . Magazine (Tokyo) 100 : 17 – 35 . Furness , C. A. , and P. J. Rudall . 2001 . The tapetum in basal angio- Jaramillo , M. A. , and P. S. Manos . 2001 . Phylogeny and patterns of sperms: Early diversity. International Journal of Plant Sciences 162 : fl oral diversity in the genus Piper (Piperaceae). American Journal of 375 – 392 . Botany 88 : 706 – 716 . Gabarayeva , N. I. , V. V. Grigorjeva , and J. R. Rowley . Jaramillo , M. A. , P. S. Manos , and E. A. Zimmer . 2004 . Phylogenetic 2003 . Sporoderm ontogeny in Cabomba aquatica (Cabombaceae). relationships of the perianthless Piperales: Reconstructing the evolu- Review of Palaeobotany and Palynology 127 : 147 – 173 . tion of fl oral development. International Journal of Plant Sciences Gilg , E. 1925 . Canellaceae. In A. Engler, and K. Prantl [eds.], Die nat ü r- 165 : 403 – 416 . lichen Pfl anzenfamilien, 2nd ed., vol. 21, 323– 328. Engelmann, Jordan , G. J. , R. A. Dillon , and P. H. Weston . 2005 . Solar radiation as Leipzig, Germany. a factor in the evolution of scleromorphic leaf anatomy in Proteaceae. Gonz á lez , F. 1999 . Infl orescence morphology and the systematics of American Journal of Botany 92 : 789 – 796 . Aristolochiaceae. Systematics and Geography of Plants 68 : 159 – 172 . Junell , S. 1931 . Die Entwicklungsgeschichte von Circaeaster agrestis. Gonz á lez , F. , and P. Rudall . 2001 . The questionable affi nities of Lactoris : Svensk Botanisk Tidskrift 25 : 238 – 270 . Evidence from branching pattern, infl orescence morphology, and stipule Kamelina , O. P. 1997 . An addition to the embryology of Lactoridaceae development. American Journal of Botany 88 : 2143 – 2150 . and Fouquieriaceae. Botanicheskii Zhurnal 82 : 25 – 29 . Grayum , M. H. 1992 . Comparative external pollen structure of the Araceae Karol , K. G. , Y. Suh , G. E. Schatz , and E. A. Zimmer . 2000 . Molecular and putatively related taxa. Monographs in Systematic Botany from evidence for the phylogenetic position of Takhtajania in the the Missouri Botanical Garden 43 : 1 – 167 . Winteraceae: Inference from nuclear ribosomal and chloroplast gene Halbritter , H. , and M. Hesse . 1993 . Sulcus morphology in some mono- spacer sequences. Annals of the Missouri Botanical Garden 87 : cot families. Grana 32 : 87 – 99 . 414 – 432 . Hamann , U. , K. Kaplan , and T. R ü bsamen . 1979 . Ü ber die Kelly , L. M. 1997 . A cladistic analysis of Asarum (Aristolochiaceae) and Samenschalenstruktur der Hydatellaceae (Monocotyledoneae) und implications for the evolution of herkogamy. American Journal of die systematische Stellung von Hydatella fi lamentosa. Botanische Botany 84 : 1752 – 1765 . Jahrbü cher fü r Systematik 100 : 555 – 563 . Kim , S. , C.-W. Park , Y.-D. Kim , and Y. Suh . 2001 . Phylogenetic re- Henry , A. 1847 . Knospenbilder, ein Beitrag zur Kenntnis der Laubknospen lationships in family Magnoliaceae inferred from ndhF sequences. und der Verzweigungsart der Pfl anzen. Nova Acta Academiae American Journal of Botany 88 : 717 – 728 . Caesareae Leopoldino-Carolinae 22 : 169 – 342 . Kim , Y.-D. , S.-H. Kim , C. H. Kim , and R. K. Jansen . 2004b . Phylogeny Heo , K. , Y. Kimoto , M. Riveros , and H. Tobe . 2004 . Embryology of of Berberidaceae based on sequences of the chloroplast gene ndhF. Gomortegaceae (Laurales); Characteristics and character evolution. Biochemical Systematics and Ecology 32 : 291 – 301 . Journal of Plant Research 117 : 221 – 228 . Kimoto , Y. , and H. Tobe . 2001 . Embryology of Laurales: A review and Heo , K. , and H. Tobe . 1995 . Embryology and relationships of Gyrocarpus perspectives. Journal of Plant Research 114 : 247 – 267 . and Hernandia (Hernandiaceae). Journal of Plant Research 108 : Kimoto , Y. , and H. Tobe . 2003 . Embryology of Siparunaceae (Laurales): 327 – 341 . Characteristics and character evolution. Journal of Plant Research Hoot , S. B. 1995 . Phylogeny of the Ranunculaceae based on preliminary 116 : 281 – 294 . atpB, rbcL and 18S nuclear ribosomal DNA sequence data. Plant K ö hler , E. 1981 . Pollen morphology of the West Indian — Central Systematics and Evolution ( Supplement ) 9: 241 – 251 . American species of the genus Buxus L. (Buxaceae) with reference to Hoot , S. B. , A. Culham , and P. R. Crane . 1995 . Phylogenetic relation- taxonomy. Pollen et Spores 23 : 37 – 91 . ships of Lardizabalaceae and Sargentodoxaceae: Chloroplast and Kö hler , E. , and P. Brü ckner . 1982 . Die Pollenmorphologie der nuclear DNA sequence evidence. Plant Systematics and Evolution afrikanischen Buxus - und Notobuxus -Arten (Buxaceae) und ihre ( Supplement ) 9: 195 – 199 . systematische Bedeutung. Grana 21 : 71 – 82 . Hoot , S. B. , and A. W. Douglas . 1998 . Phylogeny of the Proteaceae Kubitzki , K. 1969 . Monographie der Hernandiaceen. Botanische based on atpB and atpB-rbcL intergenic spacer region sequences. Jahrbü cher fü r Systematik 69 : 79 – 209 . Australian Systematic Botany 11 : 301 – 320 . Kubitzki , K. [ed.] 1993 . The families and genera of vascular plants, vol. 2. Hoot , S. B. , J. W. Kadereit , F. R. Blattner , K. B. Jork , A. E. Springer, Berlin, Germany. Schwarzbach , and P. R. Crane . 1997 . Data congruence and phy- Kubitzki , K. 1998 . Taccaceae. In K. Kubitzki [ed.], The families and gen- logeny of the Papaveraceae s.l. based on four data sets: atpB and era of vascular plants, vol. 3, 425– 428. Springer, Berlin, Germany. rbcL sequences, trnK restriction sites, and morphological characters. Leinfellner , W . 1962 . Ü ber die Variabilit ä t der Bl ü ten von Tofi eldia Systematic Botany 22 : 575 – 590 . calyculata . III. Zusammenfassende Ü bersicht der vorgefun- Hu , Z.-H. , and J. Yang . 1987 . Morphological studies on Circaeaster denen Abweichungen. Ö sterreichische Botanische Zeitschrift 109: agrestis Maxim. (1) Process of embryological development. Acta 395 – 430. Phytotaxonomica Sinica 25 : 350 – 356 . Leins , P. , and C. Erbar. 1996 . Early fl oral developmental studies in Hu , Z.-H. , J. Yang , R. Q. Jing , and Z. M. Dong . 1990 . Morphological Annonaceae. In W. Morawetz, and H. Winkler [eds.], Reproductive studies on Circaeaster agrestis II. Morphology and anatomy of fl ower, morphology in Annonaceae, 1– 27. Biosystematics and Ecology fruit and seed. Cathaya 2 : 77 – 88 . Series 10. Ö sterreichische Akademie der Wissenschaften, Vienna, Huber , H. 1993 . Aristolochiaceae. In K. Kubitzi, J. G. Rohwer, and V. Austria. Bittrich [eds.], The families and genera of vascular plants, vol. 2, Leins , P. , C. Erbar , and W. van Heel . 1988 . Note on the fl oral develop- 129 – 137. Springer, Berlin, Germany. ment of Thottea (Aristolochiaceae). Blumea 33 : 357 – 370 . January 2009] Endress and Doyle — Ancestral flowers 65

Les , D. H. 1985 . The taxonomic signifi cance of plumule morphology in Nowicke , J. W. , and J. J. Skvarla . 1982 . Pollen morphology and the re- Ceratophyllum (Ceratophyllaceae). Systematic Botany 10 : 338 – 346 . lationships of Circaeaster, of Kingdonia, and of Sargentodoxa to the Les , D. H. 1993 . Ceratophyllaceae. In K. Kubitzki, J. G. Rohwer, and Ranunculales. American Journal of Botany 69 : 990 – 998 . V. Bittrich [eds.], The families and genera of vascular plants, vol. 2, Occhioni , P. 1994 . Cinnamodendron axillare (Nees et Mart.) Endl. 246 – 250. Springer, Berlin, Germany. (Canellaceae), esp é cie arb ó rea em perigo de extin ç ã o. Arquivos do Les , D. H. , E. L. Schneider , D. J. Padgett , P. S. Soltis , D. E. Soltis , and Jardim Botâ nico do Rio de Janeiro 32 : 107 – 115 . M. Zanis . 1999 . Phylogeny, classifi cation and fl oral evolution of water Oganezova , G. G. 1984 . Morphological and anatomical specifi c fea- lilies (Nymphaeaceae; Nymphaeales): A synthesis of non-molecular, tures of seed and fruit in some representatives of the subfamily rbcL, matK , and 18S rDNA data. Systematic Botany 24 : 28 – 46 . Melanthioideae (Liliaceae) in relation with their systematics and phy- Li , J.-H. , J. Ledger , T. Ward , and P. del Tredici . 2004 . Phylogenetics logeny. Botanicheskii Zhurnal 69 : 772 – 781 . of Calycanthaceae based on molecular and morphological data, with Oh , I.-C. , T. Denk , and E. M. Friis . 2003 . Evolution of Illicium a special reference to divergent paralogues of the nrDNA ITS region. (Illiciaceae): Mapping morphological characters on the molecular Harvard Papers in Botany 9 : 69 – 82 . tree. Plant Systematics and Evolution 240 : 175 – 209 . Liang , H.-X. 1994 . On the systematic signifi cance of fl oral organogenesis Ø rgaard , M. 1991 . The genus Cabomba (Cabombaceae) — A taxonomic in Saururaceae. Acta Phytotaxonomica Sinica 32 : 425 – 432 . study. Nordic Journal of Botany 11 : 179 – 203 . Liang , H.-X. , and S. C. Tucker . 1995 . Floral ontogeny of Zippelia Periasamy , K. 1962 . The ruminate endosperm: Development and type of begoniaefolia and its familial affi nity: Saururaceae or Piperaceae? rumination. In P. Maheshwari [ed.], Plant embryology: A symposium, American Journal of Botany 82 : 681 – 689 . 62 – 74. Council of Scientifi c and Industrial Research, New Delhi, India. Linder , H. P. , and I. K. Ferguson . 1985 . On the pollen morphology and Posluszny , U. 1983 . Re-evaluation of certain key relationships in phylogeny of the Restionales and Poales. Grana 24 : 65 – 76 . the Alismatidae: Floral organogenesis of Scheuchzeria palustris Liu , Z. , G. Hao , Y.-B. Luo , L. B. Thien , S. W. Rosso , A.-M. Lu , (Scheuchzeriaceae). American Journal of Botany 70 : 925 – 933 . and Z.-D. Chen . 2006 . Phylogeny and androecial evolution in Praglowski , J. 1979 . Winteraceae. World pollen and spore fl ora, vol. 8. Schisandraceae, inferred from sequences of nuclear ribosomal DNA Almqvist and Wiksell, Stockholm, Sweden. ITS and chloroplast DNA trnL-F regions. International Journal of Prakash , N. , D. B. Forman , and S. J. Griffith . 1984 . Gametogenesis in Plant Sciences 167 : 539 – 550 . Galbulimima belgraveana (Himantandraceae). Australian Journal of Loconte , H. 1993 . Berberidaceae. In K. Kubitzki, J. G. Rohwer, and Botany 32 : 605 – 612 . V. Bittrich [eds.], The families and genera of vascular plants, vol. 2, Prakash , N. , and B. K. Kin . 1982 . Flower and fruit development in Piper 147 – 152. Springer, Berlin, Germany. nigrum L. cv. Kuching. Malaysian Journal of Science 7 : 11 – 19 . Lyew , J. , Z. Li , L.-C. Yuan , Y.-B. Luo , and T. L. Sage . 2007 . Pollen tube Prychid , C. J. , and P. J. Rudall . 1999 . Calcium oxalate crystals in growth in association with a dry-type stigmatic transmitting tissue and monocotyledons: A review of their structure and systematics. Annals extragynoecial compitum in the basal angiosperm Kadsura longipedun- of Botany 84 : 725 – 739 . culata (Schisandraceae). American Journal of Botany 94 : 1170 – 1182 . Qin , H. N. 1997 . A taxonomic revision of the Lardizabalaceae. Cathaya Magall ó n-Puebla , S. , P. S. Herendeen , and P. R. Crane . 8-9 : 1 – 214 . 1997 . Quadriplatanus georgianus gen. et sp. nov.: Staminate and pistillate Remizova , M. , and D. Sokoloff . 2003 . Infl orescence and fl oral morphol- platanaceous fl owers from the Late Cretaceous (Coniacian-Santonian) of ogy in Tofi eldia (Tofi eldiaceae) compared with Araceae, Acoraceae Georgia, U.S.A. International Journal of Plant Sciences 158 : 373 – 394 . and Alismatales s.str. Botanische Jahrbü cher fü r Systematik 124 : Markgraf , F. 1981 . Abteilung Angiospermae, Bedecktsamige Pfl anzen. 255 – 271 . In G. Hegi [ed.], Illustrierte Flora von Mitteleuropa, 3rd ed., vol. I, 2, Remizowa , M. V. , D. D. Sokoloff , and P. J. Rudall . 2006 . Evolution 149 – 260. Parey, Berlin, Germany. of the monocot gynoecium: Evidence from comparative morphol- Meng , S.-W. , Z.-D. Chen , D.-Z. Li , and H.-X. Liang . 2002 . Phylogeny ogy and development in Tofi eldia, Japanolirion, Petrosavia , and of Saururaceae based on mitochondrial matR gene sequence data. Narthecium. Plant Systematics and Evolution 258 : 183 – 209 . Journal of Plant Research 115 : 71 – 76 . Renner, S. S. 1998 . Phylogenetic affi nities of Monimiaceae based on Meng , S.-W. , A. W. Douglas , D.-Z. Li , Z.-D. Chen , H.-X. Liang , and cpDNA gene and spacer sequences. Perspectives in Plant Ecology, J.-B. Yang . 2003 . Phylogeny of Saururaceae based on morphology Evolution and Systematics 1: 61 – 77. and fi ve regions from three plant genomes. Annals of the Missouri Renner , S. S. 2004 . Variation in diversity among Laurales, Early Botanical Garden 90 : 592 – 602 . Cretaceous to Present. Biologiske Skrifter 55: 441 – 458. Metcalfe , C. R. 1987 . Anatomy of the dicotyledons, vol. 3, 2nd ed. Renner , S. S. , D. B. Foreman , and D. Murray . 2000 . Timing transan- Clarendon Press, Oxford, UK. tarctic disjunctions in the Atherospermataceae (Laurales): Evidence Metcalfe , C. R. , and L. Chalk . 1950 . Anatomy of the dicotyledons. from coding and noncoding chloroplast sequences. Systematic Biology Clarendon Press, Oxford, UK. 49 : 579 – 591 . Moseley , M. F. Jr ., I. J. Mehta , P. S. Williamson , and H. Kosakai . Renner , S. S. , and G. Hausner . 2005 . Siparunaceae. Flora Neotropica 1984 . Morphological studies of the Nymphaeaceae (sensu lato). Monographs 95 : 1 – 247 . XIII. Contributions to the vegetative and fl oral structure of Cabomba. Renner , S. S. , A. E. Schwarzbach , and L. Lohmann . 1997 . Phylogenetic American Journal of Botany 71 : 902 – 924 . position and fl oral function of Siparuna (Siparunaceae: Laurales). Murbeck , S. 1912 . Untersuchungen ü ber den Blü tenbau der Papaveraceen. International Journal of Plant Sciences 158 ( Supplement ): S89 – S98 . Kungliga Svenska Vetenskapsakademiens Handlingar 50 : 1 – 168 . Richardson , F. C. 1969 . Morphological studies of the Nymphaeaceae. Neinhuis , C. , C. Wanke , K. W. Hilu , K. M ü ller , and T. Borsch . IV. Structure and development of the fl ower of Brasenia schreberi 2005 . Phylogeny of Aristolochiaceae based on parsimony, likelihood, Gmel. University of California Publications in Botany 47 : 1 – 101 . and Bayesian analyses of trnL-trnF sequences. Plant Systematics and Richardson , J. E. , L. W. Chatrou , J. B. Mols , R. H. J. Erkens , and M. Evolution 250 : 7 – 26 . D. Pirie. 2004 . Historical biogeography of two cosmopolitan fami- Nikiticheva , Z. I. , and O. B. Proskurina . 1992 . Embryology of lies of fl owering plants: Annonaceae and Rhamnaceae. Philosophical Scheuchzeria palustris (Scheuchzeriaceae). Botanicheskii Zhurnal Transactions of the Royal Society of London, B, Biological Sciences 77 : 3 – 18 . 359 : 1495 – 1508 . Nowicke , J. W. , and J. J. Skvarla . 1979 . Pollen morphology: The po- Rudall , P. J. , and M. Buzgo . 2002 . Evolutionary history of the monocot tential infl uence in higher order systematics. Annals of the Missouri leaf. In Q. C. B. Cronk, R. M. Bateman, and J. A. Hawkins [eds.], Botanical Garden 66 : 633 – 700 . Developmental genetics and plant evolution, 431– 458. Taylor & Nowicke , J. W. , and J. J. Skvarla . 1981 . Pollen morphology and phylo- Francis, London, UK. genetic relationships of the Berberidaceae. Smithsonian Contributions Rudall , P. J. , and C. A. Furness . 1997 . Systematics of Acorus : Ovule to Botany 50 : 1 – 83 . and anther. International Journal of Plant Sciences 158 : 640 – 651 . 66 American Journal of Botany

Rutishauser , R. , and R. Sattler . 1987 . Complementarity and heuristic Tucker , S. C. , A. W. Douglas , and H.-X. Liang . 1993 . Utility of on- value of contrasting models in structural botany. II. Case studies on togenetic and conventional characters in determining phylogenetic leaf whorls: Equisetum and Ceratophyllum. Botanische Jahrbü cher relationships of Saururaceae and Piperaceae (Piperales). Systematic fü r Systematik 109 : 227 – 255 . Botany 18 : 614 – 641 . Sampson , F. B. 2000a . Pollen diversity in some modern magnoli- Utech , F. H. 1978 . Floral vascular anatomy of the monotypic Japanese ids. International Journal of Plant Sciences 161 ( Supplement ): Metanarthecium luteoviride Maxim. (Liliaceae-Melanthoideae). S193 – S210 . Annals of the Carnegie Museum 47 : 455 – 477 . Sampson , F. B. 2000b . The pollen of Takhtajania perrieri (Winteraceae). Venkata Rao , C. 1960 . Studies in the Proteaceae I. Tribe Persoonieae. Annals of the Missouri Botanical Garden 87 : 380 – 388 . Proceedings of the National Institute of Science of India B 26: 300-337. Saunders , R. M. K. 1998 . Monograph of Kadsura (Schisandraceae). Venkata Rao , C. 1961 . Studies in the Proteaceae II. Tribes Placospermeae Systematic Botany Monographs 54 : 1 – 106 . and Conospermeae. Proceedings of the National Institute of Science Sauquet, H., 2003 . Androecium diversity and evolution in Myristicaceae of India B 27: 126-151. (Magnoliales), with a description of a new Malagasy genus, Doyleanthus Venkata Rao , C. 1971 . Proteaceae. Council of Scientifi c and Industrial gen. nov. American Journal of Botany 90: 1293– 1305. Research, New Delhi, India. Schneider , E. L. , and S. Carlquist . 1996 . Conductive tissue in Vink , W. 1970 . The Winteraceae of the Old World I. Pseudowintera and Ceratophyllum demersum (Ceratophyllaceae). Sida 17 : 437 – 443 . Drimys , morphology and taxonomy. Blumea 18 : 225 – 354 . Sch ö ffel , K. 1932 . Untersuchungen ü ber den Bl ü tenbau der Vogel , S. 1998 . Remarkable nectaries: Structure, ecology, organophyl- Ranunculaceen. Planta 17 : 315 – 371 . etic perspectives II. Nectarioles. Flora 193 : 1 – 29 . Schols , P. , C. A. Furness , V. Merckx , P. Wilkin , and E. Smets . von Balthazar , M. , P. K. Endress , and Y.-L. Qiu . 2000 . Phylogenetic 2005 . Comparative pollen development in Dioscoreales. International relationships in Buxaceae based on nuclear ITS and plastid ndhF Journal of Plant Sciences 166 : 909 – 924 . sequences. International Journal of Plant Sciences 161 : 785 – 792 . Sch ö nland , S. 1883 . Ü ber die Entwicklung der Bl ü ten und Frucht bei den Walker , J. W. 1976a . Comparative pollen morphology and phylogeny of the Platanen. Botanische Jahrbü cher f ü r Systematik 4 : 308 – 327 . ranalean complex. In C. B. Beck [ed.], Origin and early evolution of an- Schulze , W. 1978 . Beitr ä ge zur Taxonomie der Liliifl oren. IV. Melanthiaceae. giosperms, 241 – 299. Columbia University Press, New York, USA. Wissenschaftliche Zeitschrift der Friedrich-Schiller Universitä t Jena . Walker , J. W. 1976b . Evolutionary signifi cance of the exine in the pollen of Mathematische-Naturwissenschaftliche Reihe 27 : 87 – 95 . primitive angiosperms. In I. K. Ferguson, and J. Muller [eds.], The evolu- Shamrov , I. I. 1983a . Anthecological investigation of three species of the tionary signifi cance of the exine, 251– 308. Academic Press, London, UK. genus Ceratophyllum (Ceratophyllaceae). Botanicheskii Zhurnal 68 : Wang , F. , D. Z. Li , and J. B. Yang . 2002 . Molecular phylogeny of the 1357 – 1365 . Lardizabalaceae based on trnL-F sequences and combined chloroplast Shamrov , I. I. 1983b . The structure of the anther and some peculiar data. Acta Botanica Sinica 44 : 971 – 977 . features of the microsporogenesis and pollen grain development in Wang , W. , Z.-D. Chen , Y. Liu , R.-Q. Li , and J.-H. Li . 2007 . Phylogenetic the representatives of the genus Ceratophyllum (Ceratophyllaceae). and biogeographic diversifi cation of Berberidaceae in the northern Botanicheskii Zhurnal 68 : 1662 – 1667 . hemisphere. Systematic Botany 32 : 731 – 742 . Staedler , Y. M. , P. H. Weston , and P. K. Endress . 2009 . Gynoecium Williamson , P. S. , and E. L. Schneider . 1994 . Floral aspects of structure and development in Calycanthaceae (Laurales). International Barclaya (Nymphaeaceae): Pollination, ontogeny and structure. Plant Journal of Plant Sciences 170: in press . Systematics and Evolution ( Supplement ) 8: 159 – 173 . Steyermark , J. A. 1971 . Notes on the genus Sarcorhachis Trel. Wilson , T. K. 1966 . The comparative morphology of the Canellaceae. (Piperaceae). Pittieria 1971(3): 29 – 38. IV. Floral morphology and conclusions. American Journal of Botany Takahashi , M. , and S. Kawano . 1989 . Pollen morphology of the 53 : 336 – 343 . Melanthiaceae and its systematic implications. Annals of the Missouri Wu , H.-C. , H.-J. Su , and J.-M. Hu . 2007 . The identifi cation of A-, B-, C-, Botanical Garden 76 : 863 – 876 . and E-class MADS-box genes and implications for perianth evolution Takhtajan , A. [ed.] 1985 . Anatomia seminum comparativa in the basal eudicot Trochodendron aralioides (Trochodendraceae). [Sravnitel’ naya anatomiya semyan], vol. 1. Nauka, Leningrad, USSR International Journal of Plant Sciences 168 : 775 – 799 . [St. Petersburg, Russia] . Wunderlich , R. 1936 . Vergleichende Untersuchungen von Pollenk ö rnern Takhtajan , A. [ed.] 1988 . Anatomia seminum comparativa, vol. 2. Nauka, einiger Liliaceen und Amaryllidaceen. Ö sterreichische Botanische Leningrad, USSR [St. Petersburg, Russia] . Zeitschrift 85: 32 – 55. Takhtajan , A. [ed.] 1991 . Anatomia seminum comparativa, vol. 3. Nauka, Yakovlev , M. S. [ed.] 1981 . Comparative embryology of fl owering plants Leningrad, USSR [St. Petersburg, Russia] . [Sravnitel ’ naya embriologiya tsvetkovykh rasteniy]. Winteraceae- Tamura , M. 1972 . Morphology and phyletic relationships of the Juglandaceae. Nauka, Leningrad, USSR [St. Petersburg, Russia] . Glaucidiaceae. Botanical Magazine (Tokyo) 85 : 29 – 41 . Yakovlev , M. S. [ed.] 1990 . Comparative embryology of fl ower- Tamura , M. 1982 . Relationship of Barclaya and classifi cation of ing plants. Butomaceae-Lemnaceae. Nauka, Leningrad, USSR [St. Nymphaeales. Acta Phytotaxonomica Geobotanica 33 : 336 – 345 . Petersburg, Russia] . Tamura , M. 1995 . Ranunculaceae. In A. Engler, K. Prantl, and P. Hiepko Zavada , M. S. 1983 . Comparative morphology of monocot pollen and [eds.], Die nat ü rlichen Pfl anzenfamilien (2nd ed.), vol. 17a, IV, 1 – evolutionary trends of apertures and wall structures. Botanical Review 555. Duncker & Humblot, Berlin, Germany. 49 : 331 – 379 . Tamura , M. N. 1998 . Nartheciaceae. In K. Kubitzki [ed.], The families and Zhang , L.-B. , and S. S. Renner . 2003 . The deepest splits in Chloranthaceae genera of vascular plants, vol. 3, 381 – 392. Springer, Berlin, Germany. as resolved by chloroplast sequences. International Journal of Plant Terabayashi , S. 1983 . Studies in the morphology and systematics of Sciences 164 ( Supplement ): S383 – S392 . Berberidaceae. VII. Floral anatomy of Nandina domestica Thunb. Zomlefer , W. B. 1997a . The genera of Melanthiaceae in the southeastern Journal of Phytogeography and Taxonomy 31 : 16 – 21 . United States. Harvard Papers in Botany 2 : 133 – 177 . Tian , X., L. Zhang , and Y. Ren . 2006 . Development of fl owers and Zomlefer , W. B. 1997b . The genera of Nartheciaceae in the southeastern infl orescences of Circaeaster (Circaeasteraceae, Ranunculales). Plant United States. Harvard Papers in Botany 2 : 195 – 211 . Systematics and Evolution 256 : 89 – 96 . Zomlefer , W. B. 1997c . The genera of Tofi eldiaceae in the southeastern Tobe , H. 2003 . Hydrastidaceae. In K. Kubitzki and C. Bayer [eds.], The United States. Harvard Papers in Botany 2 : 179 – 194 . families and genera of vascular plants, vol. 5, 405 – 409. Springer, Zomlefer , W. B. , N. H. Williams , W. M. Whitten , and W. S. Berlin, Germany. Judd . 2001 . Generic circumscription and relationships in the tribe Tobe , H. , and R. C. Keating . 1985 . The morphology and anatomy of Melanthieae (Liliales, Melanthiaceae), with emphasis of Zigadenus : Hydrastis (Ranunculales): Systematic reevaluation of the genus. Evidence from ITS and trnl -F sequence data. American Journal of Botanical Magazine (Tokyo) 98 : 291 – 316 . Botany 88 : 1657 – 1669 .