(Platanaceae), a Basal Eudicot1

American Journal of Botany 86(11): 1523±1537. 1999.

A DEVELOPMENTAL AND EVOLUTIONARY ANALYSIS OF EMBRYOLOGY IN PLATANUS (PLATANACEAE), A BASAL EUDICOT1

SANDRA K. FLOYD,2 VERONICA T. L ERNER, AND WILLIAM E. FRIEDMAN

Department of Environmental, Population, and Organismic Biology, Campus Box 334, University of Colorado, Boulder, Colorado 80309

The Platanaceae are an early derived eudicot lineage and therefore occupy a key position for understanding reproductive character diversi®cation associated with the early evolutionary radiation of ¯owering plants. We conducted an embryological study of Platanus racemosa in order to provide critical data on de®ning angiosperm reproductive characters for this important group. Female gametophyte development is monosporic. Embryogenesis occurs in a series of stages including zygote elongation and division, development of a linear proembryo, formation of the embryo proper, histogenesis, organogenesis, and growth. Endosperm development is a complex process that includes four distinct phases: free nuclear proliferation, cellularization of the chalazal zone, centripetal cellularization of the micropylar zone, and cellular differentiation and growth. Only the outer endosperm layer persists at seed maturity. Our ®ndings differ signi®cantly from previously published reports for Platanus, in which endosperm development was described as ab initio cellular. A comparison of endosperm development in Platanus with several closely and distantly related free nuclear taxa reveals considerable developmental variability, consistent with a hypothesis of multiple origins of free nuclear endosperm in angiosperms. Our analysis indicates that much remains to be learned about embryology in basal angiosperms. Additional developmental and comparative studies will likely reveal critical insights into the early evolution of ¯owering plants.

Key words: basal angiosperms; developmental evolution; embryology; endosperm development; eudicot; free nuclear endosperm; Platanaceae; Platanus racemosa.

Within the last two decades Darwin's ``abominable ing reproductive features of ¯owering plants have re- mystery,'' concerning the origin and early evolutionary mained virtually ignored. history of the angiosperms, has been the focus of much Although there have been a few recent embryological attention. Determining patterns of character distribution studies of basal angiosperms (Tobe et al., 1993; Heo and and evolution in basal angiosperms is critical to under- Tobe, 1995; Rudall and Furness, 1997; Svoma, 1998), standing the origin and diversi®cation of ¯owering plants much of the embryological literature describing these (Doyle and Donoghue, 1993; Friedman, 1994). However, taxa dates to the early part of this century, lacks photo- despite intense interest in the origin of ¯owering plants graphic documentation, and is fraught with inconsisten- as well as the considerable recent efforts of systematists cies and errors. In addition, important features of angio- to address questions of relationship among basal clades sperm reproductive biology, such as endosperm devel- (Donoghue and Doyle, 1989; Hamby and Zimmer, 1992; opment, have been reduced to a handful of typological Chase et al., 1993; Qiu et al., 1993; Doyle, Donoghue, categories that lack any phylogenetic context. Further- and Zimmer, 1994; Nixon et al., 1994; Hoot, MagalloÂn- more, embryological developmental patterns in basal lin- Puebla, and Crane, 1997; Qiu, 1997; Nandi, Chase, and eages have been classi®ed into types that are almost al- Endress, 1998; Soltis et al., 1998; Hoot, MagalloÂn, and ways based on the study of derived taxa. This ``top- Crane, 1999), there has been little comparative analysis down'' approach seriously limits our ability to address of the embryological characters of primitive angiosperms. fundamental questions of the origin and evolution of These include unique features such as a highly reduced these characters. As a result, existing typological schemes female gametophyte, triple fusion, and endosperm. Thus, provide little or no basis for understanding evolutionary the origin and diversi®cation of some of the most de®n- transitions between the types. An explicit goal of this study (and others in progress) is to characterize embryological development patterns in 1 Manuscript received 5 January 1999; revision accepted 16 April basal taxa (a ``bottom-up'' approach) in order to infer 1999. ontogenetic transitions that occurred during the early ra- The authors thank Sharon Swan for collecting and shipping all ¯oral materials used in this study; Dan Dvorkin for assistance with sectioning; diation of angiosperms. Indeed, a developmental and and John Herr and Andrew Douglas for thoughtful suggestions for im- phylogenetically based approach to describing and com- proving the manuscript. This work was funded by grants from the Na- paring reproductive features in primitive ¯owering plants, tional Science Foundation (DEB 9701210, IBN 9696013, BSR without a priori assumptions of typological categoriza- 9158182) and equipment grants-in-aid of research from Apple Com- tion, is essential if we are ever to make progress in solv- puter, Carl Zeiss, Compaq Computer, Fisher Scienti®c, Lasergraphics, Leica Instruments, Olympus America, and Research and Manufacturing ing Darwin's ``abominable mystery.'' Company. Recent phylogenetic analyses (Donoghue and Doyle, 2 Author for correspondence (e-mail: ¯[email protected]). 1989; Hamby and Zimmer, 1992; Chase et al., 1993; Qiu 1523 1524 AMERICAN JOURNAL OF BOTANY [Vol. 86 et al., 1993; Doyle, Donoghue, and Zimmer, 1994; Nixon et al., 1994; Hoot, MagalloÂn-Puebla, and Crane, 1997; Qiu, 1997; Nandi, Chase, and Endress, 1998; Hoot, Ma- galloÂn, and Crane, 1999) provide the following important insights into angiosperm phylogeny that help guide this analysis. Angiosperms are monophyletic. The monosul- cate Magnoliidae (magnoliids) are a nonmonophyletic basal assemblage of angiosperms from which monophyletic monocot and eudicot clades evolved. The eudicot clade includes 75% of extant ¯owering plant species (Drinnan, Crane, and Hoot, 1994). These phylogenetic ®ndings indicate that in order to understand character evolution during the early radiation of angiosperms we must focus on magnoliids, basal monocots, and basal eudicots. Platanus is the single extant genus representing one of the earliest branching eudicot clades, Platanaceae (Huf- ford and Crane, 1989; Schwarzwalder and Dilcher, 1991; Chase et al., 1993; Hoot, MagalloÂn-Puebla, and Crane, 1997; Hoot, MagalloÂn, and Crane, 1999). The platana- ceous lineage has a fossil record extending back to the early Cretaceous (Friis, Crane, and Pedersen, 1988; Friis and Crane, 1989; Friis, Pedersen, and Crane, 1994). Thus this group holds a key, transitional position in angiosperm phylogeny and is critical to understanding reproductive character diversi®cation during the origin of the largest clade of angiosperms (eudicots) from a magnoliid ances- Figs. 1±4. In¯orescences of Platanus racemosa. 1. Female in¯ores- tor. However, Platanus is also a taxon for which embry- cence with numerous female ¯owers tightly clustered on spherical head. ology is incompletely known (Johri, Ambegaokar, and Scale bar ϭ 0.5 cm. 2. Male in¯orescence prior to anthesis. Scale bar ϭ 0.5 cm. 3. Morphologically bisexual in¯orescence with both male Srivastava, 1992) and for which published reports (Brou- ¯owers releasing pollen and female ¯owers. Scale bar ϭ 0.5 cm. 4. wer, 1924; Guseinova, 1976) are contradictory. We have Higher magni®cation view of a female in¯orescence. A single ¯ower, therefore undertaken an embryological study of P. ra- consisting of nine free carpels and surrounding staminodes, is indicated cemosa in order to provide unequivocal data on de®ning by the arrow. Scale bar ϭ 0.25 cm. angiosperm reproductive characters. Our goals were (1) to provide an analysis of embryo- cally bisexual ¯owers (Fig. 3). Female ¯owers consist of seven to nine logical development in Platanus that moves beyond the free carpels surrounded by staminodes (Fig. 4) and diminutive sepals. century-old typologies that have dominated the embryological literature; and (2) to explore the evolutionary im- HistologyÐClusters of carpels were cut from in¯orescences and plications of our results. First, we report on the devel- placed into vials containing either 50 mmol/L Pipes buffer (also 5 opment of the female gametophyte (embryo sac), em- mmol/L EGTA and 1 mmol/L MgSO4) at pH 6.8, 100 mmol/L Pipes bryo, and endosperm. We then discuss the new contri- buffer (also 10 mmol/L EGTA and 2 mmol/L MgSO4) at pH 6.8, or a butions of this work and brie¯y compare our results to solution of 3:1 ethanol: acetic acid (3:1 solution). Acrolein or glutar- previous studies of Platanus. Finally, we compare Pla- aldehyde was added to the vials with Pipes buffer to a concentration of tanus embryological development with published data for 4%. Specimens were left in ®xative a minimum of 48 h, then rinsed other basal eudicots and more distantly related angio- and stored in Pipes buffer or 75% ethanol (the 3:1 ®xed specimens) at sperms in order to examine the developmental implica- 4ЊC until needed. tions of the multiple evolutionary origins of free nuclear Fixed ¯owers were dehydrated through an ethanol series to 95% eth- endosperm patterns among ¯owering plants. anol, in®ltrated with monomer A of the JB-4 embedding kit (Polysci- ences, Warrington, Pennsylvania), and embedded in an oxygen-free en- MATERIALS AND METHODS vironment. More than 1000 ovules were serially sectioned to 5-␮m thickness on either a MICROM (Walldorf, Germany) or a Leica (Nuss- CollectionsÐReproductive material from Platanus racemosa, native loch, Germany) rotary microtome using glass knives. Slides with acro- to southern California (Kaul, 1997), was harvested weekly from 17 lein or glutaraldehyde-®xed material were stained in 0.1% toluidine March 1997 through 29 July 1997 from several trees in a wild popu- blue, examined, and photographed on a Zeiss (Carl Zeiss, Jena, Ger- lation growing near Mentone, California. In addition, one dried in¯o- many) Axiophot microscope using both bright ®eld and differential in- rescence from the previous year was collected. Two voucher specimens terference contrast (DIC) optics. Slides with 3:1 ®xed material were were deposited at the University of Colorado Herbarium (COLO; Floyd stained in a solution of 0.25 ␮g/mL 4Ј,6-diamidino-2-phenylindole and Swan 97-47). In¯orescences were placed in plastic bags, kept cool, (DAPI) and 0.1 mg/mL phenylenediamine in 0.05 mol/L TRIZMA (Sig- and shipped for overnight delivery to the laboratory in Boulder, Colo- ma Chemical Co., St. Louis, Missouri) buffer, pH 7.2, for 45 min fol- rado. lowed by 5 min in a solution of 0.01% aniline blue in 0.1 mol/L TRIZ- Flowers are unisexual and clustered tightly on unisexual, spherical MA buffer to visualize sperm nuclei and callose in pollen tubes. Sec- heads (Figs. 1±2) that are arranged in a compound in¯orescence of two tions were examined and photographed on a Zeiss Axiophot microscope to seven heads on a peduncle. The plants are monoecious and are wind equipped with epi¯uorescence (HBO 50 W burner; Carl Zeiss, Jena, pollinated. Occasionally an entire in¯orescence will have morphologi- Germany). November 1999] FLOYD ET AL.ÐEMBRYOLOGY OF PLATANUS 1525

Fig. 5. Timeline of female gametophyte, endosperm, and embryo development in Platanus racemosa during the course of this study (20 wk) in relation to pollination and fertilization. Figure Abbreviations: 2-N, two-nucleate female gametophyte; 4-N, four-nucleate female gametophyte; ENDO, endosperm; ES-fert, fertilized female gametophyte; free-N, free nuclear; GPT, female gametophyte; MSC, megasporocyte; MSP, megaspore; PEN, primary endosperm nucleus.

RESULTS Basic phenologyÐA summary of events is shown in Fig. 5. Male ¯owers released pollen during the ®rst week of collection. Therefore, time zero represents anthesis/ pollination, and developmental stages are measured in weeks after anthesis. Pollen tubes were present in styles and ovaries within days of anthesis. Ovules were only rudimentary and lacked fully developed integuments at this stage. Stages of meiosis and tetrads of megaspores were observed in ovules collected 3 wk after anthesis. Mature female gametophytes were present at week 5. Early endosperm development was ®rst observed at 6 wk, indicating that fertilization takes place around week 5. Free nuclear endosperm was evident between weeks 7 and 9. Early stages of endosperm cellularization were observed at 10 wk as were early linear proembryos. Glob- ular proembryos and the ®rst stage of completely cellular endosperm were observed from specimens collected 13 wk after anthesis. Further stages of cellularization and embryo development were observed from materials collected between weeks 14 and 19 after anthesis. The embryos at this stage were about half the length of the endosperm. Embryos occupy most of the seed volume in mature fruits.

Megasporogenesis and female gametophyte develop- mentÐEach carpel contains one (rarely two) orthotropous, pendant ovules (Fig. 6). The ovule is bitegmic and crassinucellate. The micropyle is formed only by the inner integument (Fig. 7). Figs. 6±7. Light micrographs of longitudinal sections through car- A large megasporocyte (ϳ50 ␮m long) differentiates pels of Platanus racemosa, oriented with chalazal end toward top of deep within the nucellus (Fig. 8) 2 wk after pollination page. Scale bars ϭ 100 ␮m. Figure Abbreviations: CH, chalaza; GPT, and meiosis (Figs. 9, 10) results in a linear tetrad of female gametophyte; II, inner integument; M, micropyle; N, nucellus; megaspores (Fig. 11). The chalazal-most spore, always OI, outer integument; OV, ovule; ST, style. 6. Young carpel collected larger and more vacuolate than the other three, develops in week 1 prior to megasporocyte stage with pendant, orthotropous into the functional megaspore. The other three mega- ovule showing style, ovule, nucellus, inner integument closed around tip of nucellus, outer integument shorter than inner integument. 7. Later spores are small, cytoplasmically dense, and they quickly ovule with mature female gametophyte. Outer integument shorter than degenerate (Figs. 11, 12). Thus, female gametophyte de- inner integument, which alone forms the micropyle. velopment is monosporic. 1526 AMERICAN JOURNAL OF BOTANY [Vol. 86

Figs. 8±15. Light micrographs illustrating megasporocyte through four-nucleate female gametophyte stages in Platanus racemosa. All sections are longitudinal and oriented with chalazal end toward top of page. Scale bars ϭ 10 ␮m. Figure Abbreviations: MSP, functional megaspore; N, female gametophyte nucleus; V, vacuole. 8. Megasporocyte. 9. Meiosis I (metaphase) of megasporocyte. 10. Early telophase of meiosis I of megasporocyte. Developing cell plate present (arrow). 11. Linear tetrad of megaspores with chalazal functional megaspore and three degenerative megaspores. 12. Functional megaspore polarized with nucleus toward micropyle and vacuole toward chalaza. 13. Two-nucleate female gametophyte just after mitosis. 14. Later two-nucleate stage. Female gametophyte has elongated, nuclei have migrated to micropylar and chalazal ends. Chalazal nucleus is positioned between large central vacuole and smaller chalazal vacuole. 15. Four-nucleate female gametophyte, structurally similar to two nucleate stage, but with one pair of nuclei at chalazal end, between vacuoles, and one pair at extreme micropylar end.

After the nucleus of the functional megaspore under- closer to the egg apparatus than the middle of the female goes its ®rst mitotic division (Fig. 13), the daughter nu- gametophyte. The three antipodal cells are large and con- clei migrate to opposite ends of the young female ga- spicuously vacuolate (Fig. 17). After fertilization the an- metophyte. A large vacuole occupies the region between tipodal cells enlarge, stain more densely, and the nuclei the two nuclei. A smaller vacuole forms at the extreme become irregular in shape. chalazal end of the developing female gametophyte (Fig. Within the egg apparatus of the female gametophyte, 14). The female gametophyte enlarges to ϳ100 ␮min the synergids are densely cytoplasmic and exhibit an ob- length before the second mitotic division occurs. At the vious ®liform apparatus (Fig. 19). The egg cell protrudes four-nucleate stage (Fig. 15) it is similar in size and cy- somewhat farther into the female gametophyte, and its toplasmic appearance to the two-nucleate stage (Fig. 14). nucleus is usually centrally positioned (Fig. 20). In re- The female gametophyte continues to grow, and by the cently fertilized ovules one synergid is collapsed and the time it has differentiated into an eight-nucleate, seven- other intact, but it is not known whether the degenerate celled structure it is 300±400 ␮m in length (Fig. 16). synergid breaks down prior to fertilization. Following fer- In the mature cellular female gametophyte the two po- tilization, the female gametophyte elongates to 500±800 lar nuclei meet midway between the chalazal end and the ␮m before division of the primary endosperm nucleus micropylar end and fuse prior to fertilization (Figs. 16± occurs. 20). The resulting fusion nucleus was never observed Early embryogenesisÐThe zygote remains undivided November 1999] FLOYD ET AL.ÐEMBRYOLOGY OF PLATANUS 1527

and apical cells divide at least once transversely. How- ever, stages of mitosis in the two-celled proembryo were never observed. De®nition of the globular proembryo and suspensor begins with vertical cell divisions of the one or two most apical cells of the linear proembryo (Fig. 25). This produces a small, globular proembryo and a two-to-four- celled uniseriate suspensor. A variable sequence of additional vertical and transverse divisions is associated with further development of the three-dimensional globular proembryo (Fig. 26). The two-to-four-celled suspensor is usually persistent through the heart-shaped embryo stage (Fig. 27), and occasionally later (Fig. 28). However, it remains unchanged throughout embryogenesis and does not serve to push the embryo proper into the endosperm. Embryos often become dislodged and are displaced from the micropylar end of the endosperm. Approxi- mately 25% of the globular proembryos observed were located in the center of the early cellular endosperm (Fig. 29). All embryos ultimately produce a shoot apex, a root apex, and two well-developed cotyledons (Fig. 28). At maturity the embryo ®lls most of the cavity initially oc- cupied by the inner endosperm, with a thin layer of densely cytoplasmic, outer endosperm surrounding it. Cells of the mature embryo are ®lled with storage products including protein bodies and lipids (determined by Sudan IV and naphthol blue-black staining).

Endosperm developmentÐEndosperm development is depicted in Fig. 30. The primary endosperm nucleus divides in the center of the female gametophyte (Figs. 30 A±B, 31±33). No permanent cell wall is formed following mitosis, although an incipient cell plate was observed between two recently separated daughter nuclei (Fig. 32). The two endosperm nuclei migrate to opposite ends of the central cell before the second mitotic division occurs. Endosperm nuclei proceed through numerous rounds of free nuclear mitosis until Ͼ1000 nuclei are formed (Figs. 30B±G, 34±37). Initially, the nuclei are evenly spaced around a large central vacuole in a thin, Figs. 16±20. Light micrographs of the mature female gametophyte of Platanus racemosa. All sections are longitudinal and are oriented parietal layer of cytoplasm (Figs. 30E, 34, 37). Differ- with chalazal end toward top of page. Figure Abbreviations: A, antip- entiation of the free nuclear endosperm eventually pro- odal cells; E, egg; F, fusion nucleus; FA, ®liform apparatus; H, hypos- duces two cytoplasmically distinct zones. Fewer than 50 tase; SYN, synergid. 16. Female gametophyte with prominent hypos- nuclei aggregate at the extreme chalazal end of the en- tase, two antipodal cells and fusion nucleus in view. Scale bar ϭ 50 dosperm. This region of endosperm is cytoplasmically ␮m. 17. Chalazal end of female gametophyte showing hypostase, elon- dense, lacks a central vacuole, and will hereafter be re- gated nucellar cells between female gametophyte and hypostase, and two antipodal cells. Antipodals are arranged with nuclei on chalazal ferred to as the ``chalazal zone'' (Figs. 30F, 35, 36). At side and vacuole at micropylar side. Scale bar ϭ 10 ␮m. 18. Fusion the same time a ``micropylar zone'' of endosperm forms nucleus. Scale bar ϭ 10 ␮m. 19. Two synergids with prominent ®liform and comprises a large central vacuole with a parietal layer apparatus. Scale bar ϭ 10 ␮m. 20. Egg cell. Scale bar ϭ 10 ␮m. of cytoplasm and nuclei. As will be seen, these two free nuclear zones exhibit very different patterns of further development. The entire endosperm continues to elongate until the endosperm begins cellularization. However, dur- during free nuclear development to ϳ900 ␮m in length. ing this time the zygote elongates toward the chalazal end Cellularization of the coenocytic endosperm begins in of the endosperm and develops a large vacuole. The zy- the chalazal zone (Figs. 30G, 38, 39). Cell walls form gote nucleus occupies the apical region of the cell (Fig. that partition all of the chalazal cytoplasm and nuclei into 21). The ®rst cell division is transverse and produces a cells that are initially multinucleate and become uninu- basal cell, which includes the vacuole, and a smaller api- cleate later. The formation of cell walls in the chalazal cal cell (Fig. 22). Transverse divisions result in a unis- zone is rapid. It is not associated with mitosis nor is it eriate, linear proembryo of four to six cells (Figs. 23, centripetal. 24). Based on comparison of two-celled proembryos with Following cellularization of the chalazal zone, anticli- four-celled proembryos, we believe that both the basal nal walls are established within the micropylar zone of 1528 AMERICAN JOURNAL OF BOTANY [Vol. 86

Figs. 21±29. Light micrographs illustrating stages of embryo development. All sections longitudinal and oriented with chalazal end toward top of page. Scale bars in Figs. 21±26 ϭ 10 ␮m; in Figs. 27±29 ϭ 30 ␮m. 21. Zygote that has begun to elongate toward the endosperm. 22. Two- celled proembryo with large and highly vacuolate basal cell and smaller apical cell. 23. Four-celled, linear proembryo. 24. Six-celled, linear proembryo. 25. Initiation of embryo proper. 26. Later globular proembryo with four-celled suspensor. 27. Early stages of organogenesis; suspensor still present. 28. Early axial embryo with developing radical and cotyledons and clearly visible primary meristematic tissue zones, still attached to suspensor. 29. Embryo that was displaced from the micropylar end and is developing in the middle with endosperm that has cellularized around it. Scale bar ϭ 100 ␮m. the endosperm. Initially these anticlinal cell walls form (Figs. 30I, 42). The cells and alveoli in this layer are of between adjacent nuclei and end freely in the coenocyte varying sizes, and the anticlinal walls are not all perpen- (Figs. 30H, 40). Anticlinal wall formation is ®rst manifest dicular to the central cell wall (Fig. 42). Cell plates were at the chalazal end of the micropylar zone and is asso- observed that formed at oblique angles to the central cell ciated with the ®nal round of mitotic divisions in the wall. This process may be responsible for the formation coenocyte. This process, including nuclear division and of closed cells in the ®rst layer of alveoli and the irregular associated cell wall formation, occurs in a wave that be- angles and sizes of the cells and alveoli. Centripetal cel- gins at the chalazal end of the micropylar zone and pro- lularization then proceeds rapidly, resulting in a com- ceeds toward the micropylar end of the endosperm. pletely compartmentalized micropylar zone of endosperm Independent anticlinal walls of the micropylar zone composed of large, irregularly shaped, thin-walled, uni- quickly fuse with adjacent walls to form uninucleate al- nucleate, vacuolate cells (Figs. 30J, 43). No consistent veoli that surround the central vacuole (Fig. 41). At the pattern of cell formation is evident in either transverse or end of this wall initiation phase, the endosperm has a longitudinal sections and regular layers of cells are not completely cellularized chalazal zone and a single layer produced in the process of centripetal cellularization. The of uninucleate open alveoli and some closed cells sur- entire endosperm, when cellularization is complete, is rounding the central vacuole of the micropylar zone ϳ4000 ␮m long. November 1999] FLOYD ET AL.ÐEMBRYOLOGY OF PLATANUS 1529

Fig. 30. Diagrammatic summary of observed stages of endosperm and early embryo development in Platanus racemosa, drawn to relative scale and oriented with chalazal end toward top of page. (A). Fertilized female gametophyte with zygote and primary endosperm nucleus. (B). Primary endosperm nucleus divides, no wall is formed, nuclei migrate apart. (C). Four-nucleate coenocytic endosperm. (D). Eight-nucleate coenocytic endosperm. (E). Many-nucleate coenocytic endosperm with nuclei arranged in thin, single layer around a large central vacuole. (F). Coenocyte has more than doubled in length, several nuclei have formed chalazal zone, zygote has divided to form two-celled proembryo. (G). Cellularization is beginning in chalazal zone and a four- celled, linear proembryo is present. (H). Cell wall formation beginning in micropylar zone at chalazal end, between recently divided free nuclei. (I). Wall formation has continued toward micropylar end, forming a single layer of alveoli and cells around the central vacuole; early three-dimensional embryo proper present. (J). Cellularization and growth have continued to form a completely cellular endosperm consisting of large, thin-walled cells; proembryo in the globular stage. (K). Cell division and differentiation have occurred unevenly in the endosperm resulting in a zone of smaller, densely staining cells around the perimeter of the endosperm (outer endosperm) and leaving a central region of larger, empty, thin-walled cells (inner endosperm); embryo has reached organogenesis stage. (L). Axial embryo begins to elongate, growing into inner endosperm, which shows signs of degradation in advance of growing embryo.

Continued development, including cell division, results shows signs of degradation in advance of the embryo's in differentiation of the micropylar zone into two layers. progress. The endosperm reaches its maximum length of The ``outer endosperm'' is a parietal layer (about ®ve or ϳ5000 ␮m (5 mm) at this stage. In the mature seed the six cells thick) of smaller, more uniformly shaped cells nucellus is reduced to a thin, crushed layer, except at the that become ®lled with protein bodies and lipids (deter- extreme chalazal and micropylar ends. The integuments mined by Sudan IV and naphthol blue black staining), develop into a thin seed coat, and only the outer endo- whereas the central ``inner endosperm'' consists of large, sperm remains, surrounding the large, well-developed vacuolate cells with no obvious storage products (Figs. embryo. 30K±L, 44). Very little starch is present in the endosperm, as evidenced by examination with cross-polarized DISCUSSION light and iodine-potassium iodide (IKI) staining. The em- There have been few previous investigations of embryo grows into the inner endosperm (Fig. 45), which bryology in Platanus (Nicoloff, 1911; Bretzler, 1924; 1530 AMERICAN JOURNAL OF BOTANY [Vol. 86

Figs. 31±37. Light micrographs showing stages of the free nuclear phase of endosperm development in Platanus racemosa. All sections are longitudinal and oriented with chalazal end toward top of page (except Fig. 37). Figure Abbreviations: A, antipodal cell; CZ, chalazal zone; MZ, micropylar zone; V, central vacuole. 31. Fertilized female gametophyte with primary endosperm nucleus in late telophase (arrow). Scale bar ϭ 50 ␮m. 32. Higher magni®cation of dividing nucleus in Fig. 1. A forming cell plate is present (arrow) between daughter nuclei, but will not form a persistent cell wall. Scale bar ϭ 10 ␮m. 33. Recently separated daughter nuclei of primary endosperm nucleus division with no cell plate. Scale bar ϭ 10 ␮m. 34. Free nuclear endosperm consisting of a parietal layer of nuclei and cytoplasm surrounding a central vacuole, zygote present. Scale bar ϭ 10 ␮m. 35. Free nuclear endosperm stage slightly later than in Fig. 34 with chalazal zone. Scale bar ϭ 10 ␮m. 36. View of chalazal zone and one persistent antipodal cell. Scale bar ϭ 20 ␮m. 37. Transverse section of ovule with free nuclear endosperm. Three endosperm nuclei in a parietal layer of cytoplasm surrounding the central vacuole. Scale bar ϭ 25 ␮m.

Brouwer, 1924; Guseinova, 1976). All report a mono- Johansen or SoueÁges) to Platanus, but prefer to describe sporic pattern of female gametophyte development, with the developmental process based on the model proposed some variability in the descriptions of size, structure, and by Kaplan and Cooke (1997). Embryogenesis in Platanus phenology. Our ®ndings are in basic agreement with proceeds through six phases: (1) zygotic polarization these previous reports of female gametophyte develop- (Fig. 21); (2) zygotic cell division (Fig. 22); (3) ®lamen- ment. Only one study claimed to have observed early tous, uniseriate growth (Figs. 23, 24); (4) differentiation endosperm, which was described as ab initio cellular into a three-dimensional (globular) proembryo and linear (Guseinova, 1976). Very little has been reported of em- suspensor (Figs. 25, 26); (5) histogenesis/organogenesis; bryogenesis in Platanus (Bretzler, 1924; Guseinova, and (6) growth of the embryo proper (Figs. 27, 28). Al- 1976). though cell division patterns do not precisely correlate with the genesis of form in the embryo, the initial divi- Platanus embryology: new contributionsÐCell division of the zygote does establish two regions of the sion patterns associated with early embryo development proembryo with distinct developmental fates. The basal are variable in P. racemosa. The same embryonic form cell will contribute only to part of the suspensor while was produced through a variable sequence of cell divi- most of the derivatives of the apical cell will form the sions in early embryogenesis. This has been noted before embryo proper. The suspensor plays almost no role in in other angiosperm taxa (Burgess, 1985; Gifford and embryogenesis, in contrast to many angiosperms. Foster, 1989; Kaplan and Cooke, 1997) and argues Although the only previous study of early endosperm against the use of embryological classi®cation systems stages in Platanus reported an ab initio cellular pattern based solely on patterns of cell division and lineage. We of development (Guseinova, 1976), we clearly observed therefore do not assign an ``embryological type'' (sensu that endosperm in P. racemosa begins with a free nuclear November 1999] FLOYD ET AL.ÐEMBRYOLOGY OF PLATANUS 1531

Figs. 38±45. Light micrographs showing stages of the cellularization and differentiation phases of endosperm development in Platanus racemosa. All sections are longitudinal and are oriented with chalazal end toward top of page. Figure Abbreviations: ANT, antipodal cell; CCZ, cellularized chalazal zone; EMB, embryo; N, endosperm nucleus. Arrows indicate developing cell walls. 38. Cell walls forming in chalazal zone. Scale bar ϭ 25 ␮m. 39. Chalazal half of ovule in which the chalazal zone has cellularized, but the micropylar zone is still coenocytic. The antipodals are large and persistent. Scale bar ϭ 100 ␮m. 40. The next phase after the chalazal zone has cellularized. Anticlinal cell walls (perpendicular to the central cell wall) forming between pairs of recently divided endosperm nuclei at the chalazal end of the micropylar zone. Scale bar ϭ 50 ␮m. 41. Early phase of endosperm alveoli in micropylar zone. Scale bar ϭ 25 ␮m. 42. Later alveolar stage, central vacuole is surrounded by a single layer of alveoli and cells. Scale bar ϭ 50 ␮m. 43. The earliest stage of completely cellular endosperm, consisting of large, thin-walled, vacuolate cells. Scale bar ϭ 100 ␮m. 44. The endosperm differentiates into an outer endosperm layer of smaller, densely stained cells and an inner endosperm of larger, vacuolate cells into which the embryo grows. The inner endosperm breaks down in advance of the growing embryo. Scale bar ϭ 100 ␮m. 45. The embryo consumes the inner endosperm and is surrounded by the outer endosperm. Scale bar ϭ 100 ␮m. phase. However, endosperm development in Platanus in- differentiation. Within each of these stages, a complex volves much more than simple free nuclear proliferation series of events occurs, involving regional zonation and followed by cellularization. Rather, it occurs in four pri- differentiation of the developing embryo-nourishing tis- mary stages: (1) free nuclear proliferation; (2) cellulari- sue. It is this complex pattern of endosperm development zation of the chalazal zone; (3) centripetal cellularization that will form the basis for much of the following dis- of the micropylar zone; and (4) cellular proliferation and cussion. 1532 AMERICAN JOURNAL OF BOTANY [Vol. 86

Fig. 46. Endosperm development coded as three character states, ab initio cellular (cellular), free nuclear, and helobial, mapped onto two published cladograms. Shaded rectangles indicate basal eudicot taxa. The most parsimonious explanation is that free nuclear endosperm development (gray) evolved independently four times in the eudicot clade from the plesiomorphic cellular state: once in a clade including Ranunculaceae, Menispermaceae, and others; once in the clade including the Fumariaceae (closely related to Papaveraceae); once in a clade including Platanaceae, Nelumbonaceae, and Proteaceae; and once in the ancestor of all remaining eudicot lineages. Implied is the independent origin of free nuclear endosperm in the monocots (represented by Acorus on Tree A). Tree A based on Fig. 4B of Chase et al. (1993). Tree B based on the consensus tree of 15 shortest trees from the analysis of Hoot, MagalloÂn, and Crane (1999).

Evolutionary implications: multiple origins of free angiosperms in that it has a free nuclear phase of devel- nuclear endospermÐThe objective of our work is not opment. only to describe embryological development in Platanus, Free nuclear endosperm is the most common devel- but to use this information to examine and interpret the opmental type reported among angiosperms (Dahlgren, origin and evolution of de®ning angiosperm reproductive 1991; Johri, Ambegaokar, and Srivastava, 1992), and it characters such as endosperm. Our results show that free is generally discussed within the traditional typological nuclear endosperm cannot be viewed as ``a relatively framework as if it were a single phenomenon with a few simple and amorphous tissue marked by the presence of unusual variants (Vijayaraghavan and Prabhakar, 1984; only a few differentiated cell types'' (Raghavan, 1997, p. Johri, Ambegaokar, and Srivastava, 1992). However, 322) as is sometimes mistakenly claimed in the literature. when cellular, free nuclear, and helobial types are mapped We have chosen to focus on endosperm development be- as character states onto published cladograms that resolve cause it is a complex process with several distinct stages relationships among basal eudicot lineages (Chase et al. that provide many points of comparison. 1993; Hoot, MagalloÂn, and Crane, 1999), parsimonious Ab initio cellular endosperm is the most common de- character optimization indicates that free nuclear endo- velopmental pattern among basal angiosperm lineages sperm has evolved three times independently among the and phylogenetically based analyses of character distri- lower eudicots and once in the ancestor of higher eudi- bution indicate that it almost certainly represents the ple- cots (Fig. 46). This implies that free nuclear endosperm siomorphic condition in ¯owering plants (Donoghue and in the monocots was also derived independently. Multiple Scheiner, 1992; Friedman, 1992). Platanus endosperm is origins of free nuclear endosperm development among clearly derived relative to the plesiomorphic condition in dicots and monocots were also predicted by Dahlgren November 1999] FLOYD ET AL.ÐEMBRYOLOGY OF PLATANUS 1533

TABLE 1. Comparison of selected features of free nuclear endosperm development in Platanus and seven other taxa (based on literature review), including three other basal eudicot taxa, Bellendena, Papaver, and Ranunculus. Some information not available (?).

Fertilization to Centripetal No. free Chalazal complete Initiation point cellularization Embryo stage at Endopserm Taxon nuclei zone cellularization of cellularization pattern wall initiation storage product Platanus 1000s present 2mo chalazal irregular zygote-®lamentous protein, oil Bellendenaa many present ? micropylar ? ? ? Papaverb 100s ? 1wk micropylar ? ®lamentous protein, oil Ranunculusc 100s ? 2wk chalazal regular early globular protein, oil Stellariad many present ? micropylar irregular heart-shaped ? Legumese many ? 2wk micropylar irregular globular protein, oil or none Helianthusf 6±8 ? ? micropylar irregular globular protein, oil Cerealsg 1000s ? 1wk micropylar regular globular starch, protein a Venkata Rao, 1967. b Olson, 1981; Johri, Ambegaokar, and Srivastava, 1992. c Chitralekha and Bhandari, 1993; XuHan and Van Lammeren, 1993, 1997. d Newcomb, 1973. e Yeung and Cavey, 1988; Chamberlin, 1994; XuHan and Van Lammeren, 1994; Algan and Bakar, 1996. f Newcomb, 1973; Johri, Ambegaokar, and Srivastava, 1992. g Fineran, Wild, and Ingerfeld, 1981; Van Lammeren, 1988; Olsen, Potter, and Kalla, 1992; Olsen, Brown, and Lemmon, 1995; Brown, Lemmon, and Olsen, 1996.

(1991). Given this hypothesis of multiple origins of free han, 1970; Batygina, Kolesova, and Vasiljeva, 1983). En- nuclear endosperm development, we would expect to ®nd dosperm development was described as both free nuclear variability among taxa representing independent origins (Khanna, 1965; Padmanabhan, 1970) and ab initio cel- and greater similarity among taxa that share a common lular (Batygina, Kolesova, and Vasiljeva, 1983), demon- origin of free nuclear endosperm. strating the need for thorough reinvestigation of this im- Before comparing aspects of endosperm and embryo portant basal taxon. development among taxa we will review some of the key More work has been done on the Proteaceae (Kausik, embryological features of P. racemosa. The ovule is or- 1938a, b, 1941; Venkata Rao, 1965, 1967, 1969, and oth- thotropous and bitegmic (Figs. 6, 7), and the micropyle ers), although largely with genera such as Grevillea is formed by the inner integument only (Fig. 7). The ®rst which appear to be nested well within the family (Hoot phase of endosperm development involves free nuclear and Douglas, 1998) and exhibit highly derived endo- proliferation of the primary endosperm nucleus to pro- sperm developmental patterns (Venkata Rao, 1967). All duce thousands of free nuclei (Figs. 30±37). Cellulari- members of the Proteaceae exhibit free nuclear endo- zation of the coenocyte occurs ®rst in the chalazal zone, sperm development (Johri, Ambegaokar, and Srivastava, beginning when the embryo is in a ®lamentous phase, 1992). While derived taxa have been studied extensively, and initially yields a set of multinucleate cells (Figs. 5, there is a single report of endosperm development in the 30, 38, 39). The coenocytic micropylar zone then cellu- basal genus Bellendena (Hoot and Douglas, 1998). This larizes centripetally (Figs. 30, 40±43), following a ®nal taxon shares many embryological features with Platanus: mitotic wave that moves from the chalazal to the micro- an orthotropous ovule, micropyle formed by the inner pylar end. Cellularization is complete ϳ2 mo after fertil- integument only, distinct chalazal and micropylar zones ization (Fig. 5). Centripetal cellularization does not pro- in the free nuclear endosperm, and a large embryo in the duce regular layers of cells, but appears to be irregular mature seed (Venkata Rao, 1967). However, Bellendena (Fig. 43). The cellular endosperm differentiates into inner reportedly has micropylar-to-chalazal initiation of endo- and outer layers (Figs. 30, 44). The embryo grows into sperm cellularization (Venkata Rao, 1967), the opposite and replaces the inner endosperm, whereas the outer en- of Platanus. The similarities between Platanus and Bel- dosperm persists and stores protein and oil at seed ma- lendena are consistent with the hypothesis that they share turity (Figs. 30, 45). Most of these features are summa- a single evolutionary origin of free nuclear endosperm in rized in Table 1. a common ancestor. A more complete analysis of endo- Of immediate interest is the clade that includes Pla- sperm development in basal Proteaceae and Nelumbo is tanus, Nelumbo, and Proteaceae (Fig. 46). Close relation- needed to provide a broader basis for comparison within ship of these three taxa has been detected in a number of this intriguing clade. It is also clear that comparative em- recent phylogenetic analyses (Chase et al., 1993; Drin- bryology has great potential to address questions of re- nan, Crane, and Hoot, 1994; Hoot, MagalloÂn-Puebla, and lationship in the Platanaceae/Proteaceae/Nelumbonaceae Crane, 1997; Soltis et al., 1998; Stevenson and Douglas, clade, which is otherwise dif®cult given the highly spe- 1998; Hoot, MagalloÂn, and Crane, 1999), a surprising cialized and apomorphic nature of the sporophyte phase result given the morphological disparity of the three in these three families. groups. If this hypothesis of relationship is correct, a rea- Few developmental analyses of endosperm are avail- sonable prediction would be the presence of similar, evo- able for other basal eudicot taxa. However, several recent lutionarily homologous patterns of endosperm develop- reports describe aspects of endosperm development for ment in these three taxa. two taxa in the other two putative ``free nuclear'' basal Incomplete and con¯icting embryological reports have eudicot clades (Fig. 46): Ranunculus (Ranunculaceae) been published for Nelumbo (Khanna, 1965; Padmanab- (Chitralekha and Bhandari, 1993; XuHan and Van Lam- 1534 AMERICAN JOURNAL OF BOTANY [Vol. 86 meren, 1993, 1997) and Papaver (Papaveraceae) (Olson, 1981). It should be noted that neither Ranunculus nor Papaver are basal genera within Ranunculaceae and Pa- paveraceae (Hoot et al., 1997; Ro, Keener and McPheron, 1997). Our comparison is thus limited to taxa nested within the two lineages and thus with potentially more derived characteristics. Fortunately, there do not appear to be widely deviant patterns of endosperm development within these two families as has been observed in the Proteaceae (see discussion above) (Johri, Ambegaokar, and Srivastava, 1992, and references therein). In addition, the Menispermaceae represent a more basal branch in the ranunculid free nuclear clade than the Ranunculaceae (Fig. 46), yet embryological reports for taxa in the Men- ispermaceae (Sastri, 1964), although somewhat lacking in detail, appear to show congruence with descriptions for the Ranunculaceae. Thus, the use of Papaver and Ra- nunculus as representatives of the two putative free nuclear clades should provide useful information. Endosperm development in Papaver (Papaveraceae) (Olson, 1981; Johri, Ambegaokar, and Srivastava, 1992) differs in several respects (for which information is available) from Platanus and Ranunculus (Chitralekha and Bhandari, 1993; XuHan and Van Lammeren, 1993, 1997) except for the storage of protein and oil (Table 1). Pa- paver does show endosperm wall initiation at the ®lamentous embryo stage (Olson, 1981) as does Platanus, Fig. 47. Phylogenetic relationships of the families with free nuclear but differs from Platanus in some other embryological endosperm compared in the text. Each group represents a monophyletic features including having a micropyle formed by both lineage. Tree based on Chase et al. (1993). integuments and a small embryo surrounded by abundant endosperm in the mature seed (Johri, Ambegaokar, and and Peterson, 1992; Chamberlin, Horner, and Palmer, Srivastava, 1992). 1994; XuHan and Van Lammeren, 1994; Algan and Bak- Ranunculus shares some features of endosperm devel- ar, 1996), Stellaria (Caryophyllaceae) (Newcomb and opment with Platanus including chalazal-to-micropylar Fowke, 1973), and Helianthus (Asteraceae) (Newcomb, polarity of anticlinal cell wall initiation, wall initiation 1973) (Fig. 47). These three lineages all have two similar associated with mitosis, and the storage of oil and protein. features: micropylar-to-chalazal anticlinal wall initiation However, Ranunculus also differs from Platanus in the and irregular centripetal cellularization (Table 1). In gen- duration of cellularization, embryo developmental stage eral, these higher eudicot taxa have less extensive free at endosperm anticlinal wall initiation, and mode of cen- nuclear development prior to cellularization than the free tripetal wall formation. In Platanus, some anticlinal walls nuclear basal eudicots, although there is variability in this in the micropylar zone appear to converge as they grow feature. Cellularization begins when fewer than ten free in toward the central vacuole, closing some of the alveoli nuclei are present in Helianthus (Newcomb, 1973), formed when anticlinal walls are initiated (Fig. 42). Cen- whereas many free nuclei are produced before walls form tripetal cellularization continues to form cells of varying in Stellaria (Newcomb and Fowke, 1973) and legumes size and with no distinct layering (``irregular'' in Table (Algan and Bakar, 1996). Endosperm cell wall initiation 1) (Fig. 43). In contrast, anticlinal walls in Ranunculus in the higher eudicots appears to occur at a relatively grow inward without converging (Chitralekha and Bhan- advanced embryo stage, but this feature also varies dari, 1993). Periclinal walls form after rounds of mitosis among the higher eudicots. The embryo reaches the so that regular layers of cells are produced centripetally heart-shaped stage before walls are initiated in Stellaria (``regular'' in Table 1) (Chitralekha and Bhandari, 1993). (Newcomb and Fowke, 1973), but wall formation begins In addition to the characters compared in Table 1, Pla- at the globular embryo stage in legumes (Yeung and Cav- tanus embryos are large and well developed at seed ma- ey, 1988; Dute and Peterson, 1992; Chamberlin, Horner, turity and are surrounded by a thin layer of endosperm. and Palmer, 1994) and Helianthus (Newcomb, 1973). In In Ranunculus, embryos only reach an early cotyledon each of these cases, the onset of endosperm cellulariza- stage and are surrounded by copious amounts of endo- tion occurs at a much more advanced embryo stage than sperm at seed maturity (XuHan and Van Lammeren, in Platanus where the embryo is in an early ®lamentous 1997). Clearly there is variability among these three basal phase when walls begin to form in the chalazal zone (Ta- eudicot taxa (Platanus, Ranunculus, and Papaver) (Fig. ble 1). 46). Both legumes (Chamberlin, Horner, and Palmer, 1994; An even greater degree of developmental variability is Algan and Bakar, 1996) and Helianthus (Newcomb, evident among free nuclear endosperms when compari- 1973) store protein and oil, like Platanus, Ranunculus, sons are made with higher eudicot taxa, represented here and Papaver, although in some legumes the endosperm by legumes (Fabaceae) (Yeung and Cavey, 1988; Dute does not acquire any storage compounds (Chamberlin, November 1999] FLOYD ET AL.ÐEMBRYOLOGY OF PLATANUS 1535

Horner, and Palmer, 1994). There has been no report of lated, free nuclear taxa in the higher eudicots (represented endosperm storage products in Stellaria. Endosperm de- by legumes, Stellaria, and Helianthus) and monocots velopment patterns vary greatly among this sample of (represented by cereals) (Figs. 46, 47; Table 1). These higher eudicots (legumes, Helianthus, Stellaria); and they ®ndings are congruent with the hypothesis that free nu- all differ in many ways from basal eudicots (Table 1). clear endosperm has evolved independently within basal Finally, we can extend the comparison of free nuclear eudicots, higher eudicots, and monocots. endosperms to include the group most distantly related to Platanus, i.e., the monocots (Figs. 46, 47). The basal The cellular-to-free nuclear transition: developmental monocot Acorus (Chase et al., 1993; Duvall et al., 1993a, perspectiveÐAn intriguing feature observed in Platanus b; Nandi, Chase, and Endress, 1998) is reported to have was the presence of an incipient cell plate between ab initio cellular endosperm (Buell, 1938). This, along daughter nuclei derived from the primary endosperm nu- with parsimonious interpretation of character distribution cleus (Fig. 32). Although interzonal phragmoplasts that (Fig. 46), indicates that at some point during the separate fail to form cell plates have been observed during free radiations of monocot and eudicot clades from magnoliid nuclear mitosis of cereal endosperm (Olsen, Brown, and ancestors, free nuclear endosperms were also derived in- Lemmon, 1995), this is the ®rst report of this kind of dependently from the ab initio cellular pattern of devel- phenomenon in a more basal angiosperm. Both of these opment in the monocot clade. structures are suggestive of an ab initio cellular ancestry Cereal endosperm development has been well studied for free nuclear endosperm, and this is in turn consistent and characterized (Mares, Norstog, and Stone, 1975; with the hypothesis that ab initio cellular development is Morrison and O'Brien, 1976; Mares et al., 1977; Fineran, plesiomorphic for angiosperms (Donoghue and Scheiner, Wild, and Ingerfeld, 1981; Van Lammeren, 1988; Engell, 1992; Friedman, 1992). The evolutionary transition to a 1989; Bosnes, Weideman, and Olsen, 1992; Olsen, Potter, free nuclear condition would have involved a develop- and Kalla, 1992; Brown, Lemmon, and Olsen, 1994, mental disruption of normal cytokinesis. The point of dis- 1996; Olsen, Brown, and Lemmon, 1995) and exhibits a ruption appears to be different in Platanus and cereals: unique combination of features (Table 1). Extensive free in Platanus cytokinesis is interrupted at a point after cell nuclear development occurs, as in Platanus, but cellular- plate initiation but before cell wall completion; in cereals ization is completed rapidly after 1 wk rather than 2 mo cytokinesis is interrupted after phragmoplast formation (Olsen, Brown, and Lemmon, 1995). Cereals are the only but before a cell plate is formed. This again is consistent group of the eight compared in Table 1 to store abundant with the hypothesis of independent origins of free nuclear starch in the endosperm. Another unusual feature of ce- endosperm in these two groups. It would be interesting real endosperm is a regular centripetal cellularization, to know whether vestiges of cytokinesis are evident in which is shared only with Ranunculus (XuHan and Van taxa, other than Platanus and cereals, with free nuclear Lammeren, 1993) among the eight taxa compared. How- endosperm. ever, unlike Ranunculus, cereal endosperms exhibit differentiation into aleurone and starchy layers that store ConclusionsÐOur analysis of embryology in Platanus hydrolytic protein and starch, respectively (Olsen, Potter, reveals much more complexity than traditional typologi- and Kalla, 1992; Olsen, Brown, and Lemmon, 1995). cal designations suggest, particularly for endosperm. By This pattern is unique among free nuclear taxa. There are carefully examining the entire developmental process, we some similarities in the endosperm of cereals and several have observed a number of endosperm features that have other free nuclear taxa, such as micropylar-to-chalazal not been explicitly reported in basal taxa before, such as cellularization (Johri, Ambegaokar, and Srivastava, 1992) a transitory cell plate during free nuclear mitosis, chalazal (present in all taxa except Platanus and Ranunculus) and and micropylar zones with distinct modes of cellulariza- wall initiation at the globular proembryo phase (shared tion, and unique patterns of cellular differentiation. with legumes and Helianthus) (reviewed in Olsen, The initial stage of endosperm development in Platan- Brown, and Lemmon, 1995). us (free nuclear) is completely different from what was Comparison of free nuclear endosperm development previously reported (ab initio cellular). Two other basal among taxa also reveals that Platanus endosperm exhibits angiosperm taxa, Drimys winteri (Johri, Ambegaokar, and some features that are unique or at least unusual. These Srivastava, 1992) and Lactoris (Tobe et al., 1993), have include a distinct chalazal zone that cellularizes ®rst, dif- recently been shown to be ab initio cellular in contrast to ferential cellularization patterns of the chalazal and mi- earlier reports of free nuclear development. This dem- cropylar zones, a multinucleate cellular stage in the cha- onstrates the critical need for modern reevaluation of em- lazal zone, chalazal-to-micropylar cellularization, and dif- bryological characters for key basal angiosperm taxa. In- ferentiation into an inner endosperm that is consumed by correct data will lead to erroneous conclusions about the embryo and a persistent outer endosperm that is ®lled character evolution and relationship. with storage products. Comparison of embryology in Platanus with limited When features of development are compared, it is clear published data for basal Proteaceae indicates possible that free nuclear endosperms are not all the same. There evolutionary homologies, congruent with the hypothesis are differences in almost every aspect of developmental of recent shared ancestry and a common origin of free timing, patterning, and structure among the three basal nuclear endosperm development in the clade including eudicot lineages with free nuclear endosperm (represent- Platanaceae and Proteaceae. In contrast, comparison of ed by Platanus, Papaver, and Ranunculus). An even endosperm developmental characters in Platanus with greater degree of variability is evident when comparisons other free nuclear taxa reveals many signi®cant differ- with Platanus are extended to include more distantly re- ences. This is consistent with the hypothesis that free 1536 AMERICAN JOURNAL OF BOTANY [Vol. 86 nuclear endosperms have evolved numerous times within ical and ribosomal RNA data on the origin of angiosperms. Annals angiosperms, including three times during the early ra- of the Missouri Botanical Garden 81: 419±450. diation of the eudicot clade. DRINNAN, A. N., P. R. CRANE, AND S. B. HOOT. 1994. Patterns of ¯oral evolution in the early diversi®cation of non-magnoliid dicotyledons Comparative analysis of endosperm development with- (eudicots). Plant Systematics and Evolution (Supplement) 8: 93± in the appropriate phylogenetic context has the potential 122. to yield insight into reproductive character evolution, par- DUTE, R. R., AND C. M. PETERSON. 1992. Early endosperm development ticularly at the base of the eudicot clade where there is in ovules of soybean, Glycine max (L.) Merr. (Fabaceae). Annals considerable variability. However, analyses with the nec- of Botany 69: 263±271. essary level of detail are mostly restricted to phyloge- DUVALL, M. R., M. T. CLEGG,M.W.CHASE,H.G.HILLS,L.E.EGUIAR- netically derived groups such as cereals and legumes that TE,J.F.SMITH,B.S.GANT,E.A.ZIMMER, AND G. H. LEARN,JR. 1993a. Phylogenetic hypotheses for the monocotyledons construct- are distantly related to each other and to Platanus. With- ed from rbcL sequence data. Annals of the Missouri Botanical Gar- out comparable data sets for other basal taxa, a full ex- den 80: 607±619. ploration of the evolutionary history of the features we ÐÐÐ, G. H. LEARN,JR., L. E. EGUIARTE, AND M. T. CLEGG. 1993b. have reported is not possible. Future embryological in- Phylogenetic analysis of rbcL sequences identi®es Acorus calamus vestigations must include additional basal eudicots, basal as the primal extant monocotyledon. 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