A DEVELOPMENTAL AND EVOLUTIONARY ANALYSIS OF …friedmanlab.oeb.harvard.edu/files/friedmanlab/... · A DEVELOPMENTAL AND EVOLUTIONARY ANALYSIS OF EMBRYOLOGY IN PLATANUS (PLATANACEAE),

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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. LERNER, 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 reproductivecharacter diversification associated with the early evolutionary radiation of flowering plants. We conducted an embryologicalstudy of Platanus racemosa in order to provide critical data on defining angiosperm reproductive characters for this importantgroup. Female gametophyte development is monosporic. Embryogenesis occurs in a series of stages including zygoteelongation 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 findings differ significantly from previously published reportsfor Platanus, in which endosperm development was described as ab initio cellular. A comparison of endosperm developmentin 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 muchremains to be learned about embryology in basal angiosperms. Additional developmental and comparative studies will likelyreveal critical insights into the early evolution of flowering plants.

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

Within the last two decades Darwin’s ‘‘abominablemystery,’’ concerning the origin and early evolutionaryhistory of the angiosperms, has been the focus of muchattention. Determining patterns of character distributionand evolution in basal angiosperms is critical to under-standing the origin and diversification of flowering plants(Doyle and Donoghue, 1993; Friedman, 1994). However,despite intense interest in the origin of flowering plantsas well as the considerable recent efforts of systematiststo address questions of relationship among basal clades(Donoghue and Doyle, 1989; Hamby and Zimmer, 1992;Chase et al., 1993; Qiu et al., 1993; Doyle, Donoghue,and Zimmer, 1994; Nixon et al., 1994; Hoot, Magallon-Puebla, and Crane, 1997; Qiu, 1997; Nandi, Chase, andEndress, 1998; Soltis et al., 1998; Hoot, Magallon, andCrane, 1999), there has been little comparative analysisof the embryological characters of primitive angiosperms.These include unique features such as a highly reducedfemale gametophyte, triple fusion, and endosperm. Thus,the origin and diversification of some of the most defin-

1 Manuscript received 5 January 1999; revision accepted 16 April1999.

The authors thank Sharon Swan for collecting and shipping all floralmaterials used in this study; Dan Dvorkin for assistance with sectioning;and John Herr and Andrew Douglas for thoughtful suggestions for im-proving the manuscript. This work was funded by grants from the Na-tional Science Foundation (DEB 9701210, IBN 9696013, BSR9158182) and equipment grants-in-aid of research from Apple Com-puter, Carl Zeiss, Compaq Computer, Fisher Scientific, Lasergraphics,Leica Instruments, Olympus America, and Research and ManufacturingCompany.

2 Author for correspondence (e-mail: [email protected]).

ing reproductive features of flowering plants have re-mained virtually ignored.

Although there have been a few recent embryologicalstudies of basal angiosperms (Tobe et al., 1993; Heo andTobe, 1995; Rudall and Furness, 1997; Svoma, 1998),much of the embryological literature describing thesetaxa dates to the early part of this century, lacks photo-graphic documentation, and is fraught with inconsisten-cies and errors. In addition, important features of angio-sperm reproductive biology, such as endosperm devel-opment, have been reduced to a handful of typologicalcategories that lack any phylogenetic context. Further-more, embryological developmental patterns in basal lin-eages have been classified into types that are almost al-ways based on the study of derived taxa. This ‘‘top-down’’ approach seriously limits our ability to addressfundamental questions of the origin and evolution ofthese characters. As a result, existing typological schemesprovide little or no basis for understanding evolutionarytransitions between the types.

An explicit goal of this study (and others in progress)is to characterize embryological development patterns inbasal taxa (a ‘‘bottom-up’’ approach) in order to inferontogenetic transitions that occurred during the early ra-diation of angiosperms. Indeed, a developmental andphylogenetically based approach to describing and com-paring reproductive features in primitive flowering plants,without a priori assumptions of typological categoriza-tion, is essential if we are ever to make progress in solv-ing Darwin’s ‘‘abominable mystery.’’

Recent phylogenetic analyses (Donoghue and Doyle,1989; Hamby and Zimmer, 1992; Chase et al., 1993; Qiu

1524 [Vol. 86AMERICAN JOURNAL OF BOTANY

Figs. 1–4. Inflorescences of Platanus racemosa. 1. Female inflores-cence with numerous female flowers tightly clustered on spherical head.Scale bar 5 0.5 cm. 2. Male inflorescence prior to anthesis. Scale bar5 0.5 cm. 3. Morphologically bisexual inflorescence with both maleflowers releasing pollen and female flowers. Scale bar 5 0.5 cm. 4.Higher magnification view of a female inflorescence. A single flower,consisting of nine free carpels and surrounding staminodes, is indicatedby the arrow. Scale bar 5 0.25 cm.

et al., 1993; Doyle, Donoghue, and Zimmer, 1994; Nixonet al., 1994; Hoot, Magallon-Puebla, and Crane, 1997;Qiu, 1997; Nandi, Chase, and Endress, 1998; Hoot, Ma-gallon, and Crane, 1999) provide the following importantinsights into angiosperm phylogeny that help guide thisanalysis. Angiosperms are monophyletic. The monosul-cate Magnoliidae (magnoliids) are a nonmonophyleticbasal assemblage of angiosperms from which monophy-letic monocot and eudicot clades evolved. The eudicotclade includes 75% of extant flowering plant species(Drinnan, Crane, and Hoot, 1994). These phylogeneticfindings indicate that in order to understand characterevolution during the early radiation of angiosperms wemust focus on magnoliids, basal monocots, and basal eu-dicots.

Platanus is the single extant genus representing one ofthe earliest branching eudicot clades, Platanaceae (Huf-ford and Crane, 1989; Schwarzwalder and Dilcher, 1991;Chase et al., 1993; Hoot, Magallon-Puebla, and Crane,1997; Hoot, Magallon, and Crane, 1999). The platana-ceous lineage has a fossil record extending back to theearly Cretaceous (Friis, Crane, and Pedersen, 1988; Friisand Crane, 1989; Friis, Pedersen, and Crane, 1994). Thusthis group holds a key, transitional position in angiospermphylogeny and is critical to understanding reproductivecharacter diversification during the origin of the largestclade of angiosperms (eudicots) from a magnoliid ances-tor. However, Platanus is also a taxon for which embry-ology is incompletely known (Johri, Ambegaokar, andSrivastava, 1992) and for which published reports (Brou-wer, 1924; Guseinova, 1976) are contradictory. We havetherefore undertaken an embryological study of P. ra-cemosa in order to provide unequivocal data on definingangiosperm reproductive characters.

Our goals were (1) to provide an analysis of embryo-logical development in Platanus that moves beyond thecentury-old typologies that have dominated the embryo-logical literature; and (2) to explore the evolutionary im-plications of our results. First, we report on the devel-opment of the female gametophyte (embryo sac), em-bryo, and endosperm. We then discuss the new contri-butions of this work and briefly compare our results toprevious studies of Platanus. Finally, we compare Pla-tanus embryological development with published data forother basal eudicots and more distantly related angio-sperms in order to examine the developmental implica-tions of the multiple evolutionary origins of free nuclearendosperm patterns among flowering plants.

MATERIALS AND METHODS

Collections—Reproductive material from Platanus racemosa, nativeto southern California (Kaul, 1997), was harvested weekly from 17March 1997 through 29 July 1997 from several trees in a wild popu-lation growing near Mentone, California. In addition, one dried inflo-rescence from the previous year was collected. Two voucher specimenswere deposited at the University of Colorado Herbarium (COLO; Floydand Swan 97-47). Inflorescences were placed in plastic bags, kept cool,and shipped for overnight delivery to the laboratory in Boulder, Colo-rado.

Flowers are unisexual and clustered tightly on unisexual, sphericalheads (Figs. 1–2) that are arranged in a compound inflorescence of twoto seven heads on a peduncle. The plants are monoecious and are windpollinated. Occasionally an entire inflorescence will have morphologi-

cally bisexual flowers (Fig. 3). Female flowers consist of seven to ninefree carpels surrounded by staminodes (Fig. 4) and diminutive sepals.

Histology—Clusters of carpels were cut from inflorescences andplaced into vials containing either 50 mmol/L Pipes buffer (also 5mmol/L EGTA and 1 mmol/L MgSO4) at pH 6.8, 100 mmol/L Pipesbuffer (also 10 mmol/L EGTA and 2 mmol/L MgSO4) at pH 6.8, or asolution of 3:1 ethanol: acetic acid (3:1 solution). Acrolein or glutar-aldehyde was added to the vials with Pipes buffer to a concentration of4%. Specimens were left in fixative a minimum of 48 h, then rinsedand stored in Pipes buffer or 75% ethanol (the 3:1 fixed specimens) at48C until needed.

Fixed flowers were dehydrated through an ethanol series to 95% eth-anol, infiltrated with monomer A of the JB-4 embedding kit (Polysci-ences, Warrington, Pennsylvania), and embedded in an oxygen-free en-vironment. More than 1000 ovules were serially sectioned to 5-mmthickness on either a MICROM (Walldorf, Germany) or a Leica (Nuss-loch, Germany) rotary microtome using glass knives. Slides with acro-lein or glutaraldehyde-fixed material were stained in 0.1% toluidineblue, examined, and photographed on a Zeiss (Carl Zeiss, Jena, Ger-many) Axiophot microscope using both bright field and differential in-terference contrast (DIC) optics. Slides with 3:1 fixed material werestained in a solution of 0.25 mg/mL 49,6-diamidino-2-phenylindole(DAPI) and 0.1 mg/mL phenylenediamine in 0.05 mol/L TRIZMA (Sig-ma Chemical Co., St. Louis, Missouri) buffer, pH 7.2, for 45 min fol-lowed by 5 min in a solution of 0.01% aniline blue in 0.1 mol/L TRIZ-MA buffer to visualize sperm nuclei and callose in pollen tubes. Sec-tions were examined and photographed on a Zeiss Axiophot microscopeequipped with epifluorescence (HBO 50 W burner; Carl Zeiss, Jena,Germany).

November 1999] 1525FLOYD ET AL.—EMBRYOLOGY OF PLATANUS

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.

Figs. 6–7. Light micrographs of longitudinal sections through car-pels of Platanus racemosa, oriented with chalazal end toward top ofpage. Scale bars 5 100 mm. Figure Abbreviations: CH, chalaza; GPT,female gametophyte; II, inner integument; M, micropyle; N, nucellus;OI, outer integument; OV, ovule; ST, style. 6. Young carpel collectedin week 1 prior to megasporocyte stage with pendant, orthotropousovule showing style, ovule, nucellus, inner integument closed aroundtip of nucellus, outer integument shorter than inner integument. 7. Laterovule with mature female gametophyte. Outer integument shorter thaninner integument, which alone forms the micropyle.

RESULTS

Basic phenology—A summary of events is shown inFig. 5. Male flowers released pollen during the first weekof collection. Therefore, time zero represents anthesis/pollination, and developmental stages are measured inweeks after anthesis. Pollen tubes were present in stylesand ovaries within days of anthesis. Ovules were onlyrudimentary and lacked fully developed integuments atthis stage. Stages of meiosis and tetrads of megasporeswere observed in ovules collected 3 wk after anthesis.Mature female gametophytes were present at week 5.Early endosperm development was first observed at 6 wk,indicating that fertilization takes place around week 5.Free nuclear endosperm was evident between weeks 7and 9. Early stages of endosperm cellularization were ob-served at 10 wk as were early linear proembryos. Glob-ular proembryos and the first stage of completely cellularendosperm were observed from specimens collected 13wk after anthesis. Further stages of cellularization andembryo development were observed from materials col-lected between weeks 14 and 19 after anthesis. The em-bryos at this stage were about half the length of the en-dosperm. Embryos occupy most of the seed volume inmature fruits.

Megasporogenesis and female gametophyte develop-ment—Each carpel contains one (rarely two) orthotro-pous, pendant ovules (Fig. 6). The ovule is bitegmic andcrassinucellate. The micropyle is formed only by the in-ner integument (Fig. 7).

A large megasporocyte (;50 mm long) differentiatesdeep within the nucellus (Fig. 8) 2 wk after pollinationand meiosis (Figs. 9, 10) results in a linear tetrad ofmegaspores (Fig. 11). The chalazal-most spore, alwayslarger and more vacuolate than the other three, developsinto the functional megaspore. The other three mega-spores are small, cytoplasmically dense, and they quicklydegenerate (Figs. 11, 12). Thus, female gametophyte de-velopment is monosporic.


Figs. 8–15. Light micrographs illustrating megasporocyte through four-nucleate female gametophyte stages in Platanus racemosa. All sectionsare longitudinal and oriented with chalazal end toward top of page. Scale bars 5 10 mm. 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 ofmegasporocyte. Developing cell plate present (arrow). 11. Linear tetrad of megaspores with chalazal functional megaspore and three degenerativemegaspores. 12. Functional megaspore polarized with nucleus toward micropyle and vacuole toward chalaza. 13. Two-nucleate female gametophytejust after mitosis. 14. Later two-nucleate stage. Female gametophyte has elongated, nuclei have migrated to micropylar and chalazal ends. Chalazalnucleus is positioned between large central vacuole and smaller chalazal vacuole. 15. Four-nucleate female gametophyte, structurally similar to twonucleate 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-goes its first mitotic division (Fig. 13), the daughter nu-clei migrate to opposite ends of the young female ga-metophyte. A large vacuole occupies the region betweenthe two nuclei. A smaller vacuole forms at the extremechalazal end of the developing female gametophyte (Fig.14). The female gametophyte enlarges to ;100 mm inlength before the second mitotic division occurs. At thefour-nucleate stage (Fig. 15) it is similar in size and cy-toplasmic appearance to the two-nucleate stage (Fig. 14).The female gametophyte continues to grow, and by thetime it has differentiated into an eight-nucleate, seven-celled structure it is 300–400 mm in length (Fig. 16).

In the mature cellular female gametophyte the two po-lar nuclei meet midway between the chalazal end and themicropylar end and fuse prior to fertilization (Figs. 16–20). The resulting fusion nucleus was never observed

closer to the egg apparatus than the middle of the femalegametophyte. The three antipodal cells are large and con-spicuously vacuolate (Fig. 17). After fertilization the an-tipodal cells enlarge, stain more densely, and the nucleibecome irregular in shape.

Within the egg apparatus of the female gametophyte,the synergids are densely cytoplasmic and exhibit an ob-vious filiform apparatus (Fig. 19). The egg cell protrudessomewhat farther into the female gametophyte, and itsnucleus is usually centrally positioned (Fig. 20). In re-cently fertilized ovules one synergid is collapsed and theother intact, but it is not known whether the degeneratesynergid breaks down prior to fertilization. Following fer-tilization, the female gametophyte elongates to 500–800mm before division of the primary endosperm nucleusoccurs.

Early embryogenesis—The zygote remains undivided


Figs. 16–20. Light micrographs of the mature female gametophyteof Platanus racemosa. All sections are longitudinal and are orientedwith chalazal end toward top of page. Figure Abbreviations: A, antip-odal cells; E, egg; F, fusion nucleus; FA, filiform apparatus; H, hypos-tase; SYN, synergid. 16. Female gametophyte with prominent hypos-tase, two antipodal cells and fusion nucleus in view. Scale bar 5 50mm. 17. Chalazal end of female gametophyte showing hypostase, elon-gated nucellar cells between female gametophyte and hypostase, andtwo antipodal cells. Antipodals are arranged with nuclei on chalazalside and vacuole at micropylar side. Scale bar 5 10 mm. 18. Fusionnucleus. Scale bar 5 10 mm. 19. Two synergids with prominent filiformapparatus. Scale bar 5 10 mm. 20. Egg cell. Scale bar 5 10 mm.

until the endosperm begins cellularization. However, dur-ing this time the zygote elongates toward the chalazal endof the endosperm and develops a large vacuole. The zy-gote nucleus occupies the apical region of the cell (Fig.21). The first cell division is transverse and produces abasal cell, which includes the vacuole, and a smaller api-cal cell (Fig. 22). Transverse divisions result in a unis-eriate, linear proembryo of four to six cells (Figs. 23,24). Based on comparison of two-celled proembryos withfour-celled proembryos, we believe that both the basal

and apical cells divide at least once transversely. How-ever, stages of mitosis in the two-celled proembryo werenever observed.

Definition of the globular proembryo and suspensorbegins with vertical cell divisions of the one or two mostapical cells of the linear proembryo (Fig. 25). This pro-duces a small, globular proembryo and a two-to-four-celled uniseriate suspensor. A variable sequence of ad-ditional vertical and transverse divisions is associatedwith further development of the three-dimensional glob-ular proembryo (Fig. 26). The two-to-four-celled suspen-sor is usually persistent through the heart-shaped embryostage (Fig. 27), and occasionally later (Fig. 28). However,it remains unchanged throughout embryogenesis anddoes not serve to push the embryo proper into the en-dosperm.

Embryos often become dislodged and are displacedfrom the micropylar end of the endosperm. Approxi-mately 25% of the globular proembryos observed werelocated in the center of the early cellular endosperm (Fig.29). All embryos ultimately produce a shoot apex, a rootapex, and two well-developed cotyledons (Fig. 28). Atmaturity the embryo fills most of the cavity initially oc-cupied by the inner endosperm, with a thin layer ofdensely cytoplasmic, outer endosperm surrounding it.Cells of the mature embryo are filled with storage prod-ucts including protein bodies and lipids (determined bySudan IV and naphthol blue-black staining).

Endosperm development—Endosperm development isdepicted in Fig. 30. The primary endosperm nucleus di-vides in the center of the female gametophyte (Figs.30 A–B, 31–33). No permanent cell wall is formed fol-lowing mitosis, although an incipient cell plate was ob-served between two recently separated daughter nuclei(Fig. 32). The two endosperm nuclei migrate to oppositeends of the central cell before the second mitotic divisionoccurs. Endosperm nuclei proceed through numerousrounds of free nuclear mitosis until .1000 nuclei areformed (Figs. 30B–G, 34–37). Initially, the nuclei areevenly spaced around a large central vacuole in a thin,parietal layer of cytoplasm (Figs. 30E, 34, 37). Differ-entiation of the free nuclear endosperm eventually pro-duces two cytoplasmically distinct zones. Fewer than 50nuclei aggregate at the extreme chalazal end of the en-dosperm. This region of endosperm is cytoplasmicallydense, lacks a central vacuole, and will hereafter be re-ferred to as the ‘‘chalazal zone’’ (Figs. 30F, 35, 36). Atthe same time a ‘‘micropylar zone’’ of endosperm formsand comprises a large central vacuole with a parietal layerof cytoplasm and nuclei. As will be seen, these two freenuclear zones exhibit very different patterns of furtherdevelopment. The entire endosperm continues to elongateduring free nuclear development to ;900 mm in length.

Cellularization of the coenocytic endosperm begins inthe chalazal zone (Figs. 30G, 38, 39). Cell walls formthat partition all of the chalazal cytoplasm and nuclei intocells that are initially multinucleate and become uninu-cleate later. The formation of cell walls in the chalazalzone is rapid. It is not associated with mitosis nor is itcentripetal.

Following cellularization of the chalazal zone, anticli-nal walls are established within the micropylar zone of


Figs. 21–29. Light micrographs illustrating stages of embryo development. All sections longitudinal and oriented with chalazal end toward topof page. Scale bars in Figs. 21–26 5 10 mm; in Figs. 27–29 5 30 mm. 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, linearproembryo. 25. Initiation of embryo proper. 26. Later globular proembryo with four-celled suspensor. 27. Early stages of organogenesis; suspensorstill present. 28. Early axial embryo with developing radical and cotyledons and clearly visible primary meristematic tissue zones, still attached tosuspensor. 29. Embryo that was displaced from the micropylar end and is developing in the middle with endosperm that has cellularized around it.Scale bar 5 100 mm.

the endosperm. Initially these anticlinal cell walls formbetween adjacent nuclei and end freely in the coenocyte(Figs. 30H, 40). Anticlinal wall formation is first manifestat the chalazal end of the micropylar zone and is asso-ciated with the final round of mitotic divisions in thecoenocyte. This process, including nuclear division andassociated cell wall formation, occurs in a wave that be-gins at the chalazal end of the micropylar zone and pro-ceeds toward the micropylar end of the endosperm.

Independent anticlinal walls of the micropylar zonequickly fuse with adjacent walls to form uninucleate al-veoli that surround the central vacuole (Fig. 41). At theend of this wall initiation phase, the endosperm has acompletely cellularized chalazal zone and a single layerof uninucleate open alveoli and some closed cells sur-rounding the central vacuole of the micropylar zone

(Figs. 30I, 42). The cells and alveoli in this layer are ofvarying sizes, and the anticlinal walls are not all perpen-dicular to the central cell wall (Fig. 42). Cell plates wereobserved that formed at oblique angles to the central cellwall. This process may be responsible for the formationof closed cells in the first layer of alveoli and the irregularangles and sizes of the cells and alveoli. Centripetal cel-lularization then proceeds rapidly, resulting in a com-pletely compartmentalized micropylar zone of endospermcomposed of large, irregularly shaped, thin-walled, uni-nucleate, vacuolate cells (Figs. 30J, 43). No consistentpattern of cell formation is evident in either transverse orlongitudinal sections and regular layers of cells are notproduced in the process of centripetal cellularization. Theentire endosperm, when cellularization is complete, is;4000 mm long.


Fig. 30. Diagrammatic summary of observed stages of endosperm and early embryo development in Platanus racemosa, drawn to relative scaleand oriented with chalazal end toward top of page. (A). Fertilized female gametophyte with zygote and primary endosperm nucleus. (B). Primaryendosperm nucleus divides, no wall is formed, nuclei migrate apart. (C). Four-nucleate coenocytic endosperm. (D). Eight-nucleate coenocyticendosperm. (E). Many-nucleate coenocytic endosperm with nuclei arranged in thin, single layer around a large central vacuole. (F). Coenocyte hasmore than doubled in length, several nuclei have formed chalazal zone, zygote has divided to form two-celled proembryo. (G). Cellularization isbeginning 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 aroundthe central vacuole; early three-dimensional embryo proper present. (J). Cellularization and growth have continued to form a completely cellularendosperm consisting of large, thin-walled cells; proembryo in the globular stage. (K). Cell division and differentiation have occurred unevenly inthe endosperm resulting in a zone of smaller, densely staining cells around the perimeter of the endosperm (outer endosperm) and leaving a centralregion 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, resultsin differentiation of the micropylar zone into two layers.The ‘‘outer endosperm’’ is a parietal layer (about five orsix cells thick) of smaller, more uniformly shaped cellsthat become filled with protein bodies and lipids (deter-mined by Sudan IV and naphthol blue black staining),whereas the central ‘‘inner endosperm’’ consists of large,vacuolate cells with no obvious storage products (Figs.30K–L, 44). Very little starch is present in the endo-sperm, as evidenced by examination with cross-polarizedlight and iodine-potassium iodide (IKI) staining. The em-bryo grows into the inner endosperm (Fig. 45), which

shows signs of degradation in advance of the embryo’sprogress. The endosperm reaches its maximum length of;5000 mm (5 mm) at this stage. In the mature seed thenucellus is reduced to a thin, crushed layer, except at theextreme chalazal and micropylar ends. The integumentsdevelop into a thin seed coat, and only the outer endo-sperm remains, surrounding the large, well-developedembryo.

DISCUSSION

There have been few previous investigations of em-bryology in Platanus (Nicoloff, 1911; Bretzler, 1924;


Figs. 31–37. Light micrographs showing stages of the free nuclear phase of endosperm development in Platanus racemosa. All sections arelongitudinal 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 5 50mm. 32. Higher magnification of dividing nucleus in Fig. 1. A forming cell plate is present (arrow) between daughter nuclei, but will not form apersistent cell wall. Scale bar 5 10 mm. 33. Recently separated daughter nuclei of primary endosperm nucleus division with no cell plate. Scalebar 5 10 mm. 34. Free nuclear endosperm consisting of a parietal layer of nuclei and cytoplasm surrounding a central vacuole, zygote present.Scale bar 5 10 mm. 35. Free nuclear endosperm stage slightly later than in Fig. 34 with chalazal zone. Scale bar 5 10 mm. 36. View of chalazalzone and one persistent antipodal cell. Scale bar 5 20 mm. 37. Transverse section of ovule with free nuclear endosperm. Three endosperm nucleiin a parietal layer of cytoplasm surrounding the central vacuole. Scale bar 5 25 mm.

Brouwer, 1924; Guseinova, 1976). All report a mono-sporic pattern of female gametophyte development, withsome variability in the descriptions of size, structure, andphenology. Our findings are in basic agreement withthese previous reports of female gametophyte develop-ment. Only one study claimed to have observed earlyendosperm, which was described as ab initio cellular(Guseinova, 1976). Very little has been reported of em-bryogenesis in Platanus (Bretzler, 1924; Guseinova,1976).

Platanus embryology: new contributions—Cell divi-sion patterns associated with early embryo developmentare variable in P. racemosa. The same embryonic formwas produced through a variable sequence of cell divi-sions in early embryogenesis. This has been noted beforein other angiosperm taxa (Burgess, 1985; Gifford andFoster, 1989; Kaplan and Cooke, 1997) and arguesagainst the use of embryological classification systemsbased solely on patterns of cell division and lineage. Wetherefore do not assign an ‘‘embryological type’’ (sensu

Johansen or Soueges) to Platanus, but prefer to describethe developmental process based on the model proposedby Kaplan and Cooke (1997). Embryogenesis in Platanusproceeds through six phases: (1) zygotic polarization(Fig. 21); (2) zygotic cell division (Fig. 22); (3) filamen-tous, uniseriate growth (Figs. 23, 24); (4) differentiationinto a three-dimensional (globular) proembryo and linearsuspensor (Figs. 25, 26); (5) histogenesis/organogenesis;and (6) growth of the embryo proper (Figs. 27, 28). Al-though cell division patterns do not precisely correlatewith the genesis of form in the embryo, the initial divi-sion of the zygote does establish two regions of theproembryo with distinct developmental fates. The basalcell will contribute only to part of the suspensor whilemost of the derivatives of the apical cell will form theembryo proper. The suspensor plays almost no role inembryogenesis, in contrast to many angiosperms.

Although the only previous study of early endospermstages in Platanus reported an ab initio cellular patternof development (Guseinova, 1976), we clearly observedthat endosperm in P. racemosa begins with a free nuclear


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, cellularizedchalazal zone; EMB, embryo; N, endosperm nucleus. Arrows indicate developing cell walls. 38. Cell walls forming in chalazal zone. Scale bar 525 mm. 39. Chalazal half of ovule in which the chalazal zone has cellularized, but the micropylar zone is still coenocytic. The antipodals are largeand persistent. Scale bar 5 100 mm. 40. The next phase after the chalazal zone has cellularized. Anticlinal cell walls (perpendicular to the centralcell wall) forming between pairs of recently divided endosperm nuclei at the chalazal end of the micropylar zone. Scale bar 5 50 mm. 41. Earlyphase of endosperm alveoli in micropylar zone. Scale bar 5 25 mm. 42. Later alveolar stage, central vacuole is surrounded by a single layer ofalveoli and cells. Scale bar 5 50 mm. 43. The earliest stage of completely cellular endosperm, consisting of large, thin-walled, vacuolate cells.Scale bar 5 100 mm. 44. The endosperm differentiates into an outer endosperm layer of smaller, densely stained cells and an inner endosperm oflarger, vacuolate cells into which the embryo grows. The inner endosperm breaks down in advance of the growing embryo. Scale bar 5 100 mm.45. The embryo consumes the inner endosperm and is surrounded by the outer endosperm. Scale bar 5 100 mm.

phase. However, endosperm development in Platanus in-volves much more than simple free nuclear proliferationfollowed by cellularization. Rather, it occurs in four pri-mary stages: (1) free nuclear proliferation; (2) cellulari-zation of the chalazal zone; (3) centripetal cellularizationof the micropylar zone; and (4) cellular proliferation and

differentiation. Within each of these stages, a complexseries of events occurs, involving regional zonation anddifferentiation of the developing embryo-nourishing tis-sue. It is this complex pattern of endosperm developmentthat will form the basis for much of the following dis-cussion.


Fig. 46. Endosperm development coded as three character states, ab initio cellular (cellular), free nuclear, and helobial, mapped onto twopublished 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 nuclearendosperm 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 consensustree of 15 shortest trees from the analysis of Hoot, Magallon, and Crane (1999).

Evolutionary implications: multiple origins of freenuclear endosperm—The objective of our work is notonly to describe embryological development in Platanus,but to use this information to examine and interpret theorigin and evolution of defining angiosperm reproductivecharacters such as endosperm. Our results show that freenuclear endosperm cannot be viewed as ‘‘a relativelysimple and amorphous tissue marked by the presence ofonly a few differentiated cell types’’ (Raghavan, 1997, p.322) as is sometimes mistakenly claimed in the literature.We have chosen to focus on endosperm development be-cause it is a complex process with several distinct stagesthat provide many points of comparison.

Ab initio cellular endosperm is the most common de-velopmental pattern among basal angiosperm lineagesand phylogenetically based analyses of character distri-bution indicate that it almost certainly represents the ple-siomorphic condition in flowering plants (Donoghue andScheiner, 1992; Friedman, 1992). Platanus endosperm isclearly derived relative to the plesiomorphic condition in

angiosperms in that it has a free nuclear phase of devel-opment.

Free nuclear endosperm is the most common devel-opmental type reported among angiosperms (Dahlgren,1991; Johri, Ambegaokar, and Srivastava, 1992), and itis generally discussed within the traditional typologicalframework as if it were a single phenomenon with a fewunusual variants (Vijayaraghavan and Prabhakar, 1984;Johri, Ambegaokar, and Srivastava, 1992). However,when cellular, free nuclear, and helobial types are mappedas character states onto published cladograms that resolverelationships among basal eudicot lineages (Chase et al.1993; Hoot, Magallon, and Crane, 1999), parsimoniouscharacter optimization indicates that free nuclear endo-sperm has evolved three times independently among thelower eudicots and once in the ancestor of higher eudi-cots (Fig. 46). This implies that free nuclear endospermin the monocots was also derived independently. Multipleorigins of free nuclear endosperm development amongdicots and monocots were also predicted by Dahlgren


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 (?).

TaxonNo. freenuclei

Chalazalzone

Fertilization tocomplete

cellularizationInitiation point

of cellularization

Centripetalcellularization

patternEmbryo stage at

wall initiationEndopserm

storage product

PlatanusBellendenaa

Papaverb

Ranunculusc

Stellariad

Legumese

Helianthusf

Cerealsg

1000smany100s100s

manymany6–8

1000s

presentpresent

??

present???

2 mo?

1 wk2 wk

?2 wk

?1 wk

chalazalmicropylarmicropylarchalazalmicropylarmicropylarmicropylarmicropylar

irregular??

regularirregularirregularirregularregular

zygote-filamentous?

filamentousearly globularheart-shapedglobularglobularglobular

protein, oil?

protein, oilprotein, oil

?protein, oil or noneprotein, oilstarch, 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 freenuclear endosperm development, we would expect to findvariability among taxa representing independent originsand greater similarity among taxa that share a commonorigin of free nuclear endosperm.

Before comparing aspects of endosperm and embryodevelopment among taxa we will review some of the keyembryological features of P. racemosa. The ovule is or-thotropous and bitegmic (Figs. 6, 7), and the micropyleis formed by the inner integument only (Fig. 7). The firstphase of endosperm development involves free nuclearproliferation of the primary endosperm nucleus to pro-duce thousands of free nuclei (Figs. 30–37). Cellulari-zation of the coenocyte occurs first in the chalazal zone,beginning when the embryo is in a filamentous phase,and initially yields a set of multinucleate cells (Figs. 5,30, 38, 39). The coenocytic micropylar zone then cellu-larizes centripetally (Figs. 30, 40–43), following a finalmitotic wave that moves from the chalazal to the micro-pylar end. Cellularization is complete ;2 mo after fertil-ization (Fig. 5). Centripetal cellularization does not pro-duce regular layers of cells, but appears to be irregular(Fig. 43). The cellular endosperm differentiates into innerand outer layers (Figs. 30, 44). The embryo grows intoand replaces the inner endosperm, whereas the outer en-dosperm persists and stores protein and oil at seed ma-turity (Figs. 30, 45). Most of these features are summa-rized in Table 1.

Of immediate interest is the clade that includes Pla-tanus, Nelumbo, and Proteaceae (Fig. 46). Close relation-ship of these three taxa has been detected in a number ofrecent phylogenetic analyses (Chase et al., 1993; Drin-nan, Crane, and Hoot, 1994; Hoot, Magallon-Puebla, andCrane, 1997; Soltis et al., 1998; Stevenson and Douglas,1998; Hoot, Magallon, and Crane, 1999), a surprisingresult given the morphological disparity of the threegroups. If this hypothesis of relationship is correct, a rea-sonable prediction would be the presence of similar, evo-lutionarily homologous patterns of endosperm develop-ment in these three taxa.

Incomplete and conflicting embryological reports havebeen published for Nelumbo (Khanna, 1965; Padmanab-

han, 1970; Batygina, Kolesova, and Vasiljeva, 1983). En-dosperm development was described as both free nuclear(Khanna, 1965; Padmanabhan, 1970) and ab initio cel-lular (Batygina, Kolesova, and Vasiljeva, 1983), demon-strating the need for thorough reinvestigation of this im-portant basal taxon.

More work has been done on the Proteaceae (Kausik,1938a, b, 1941; Venkata Rao, 1965, 1967, 1969, and oth-ers), although largely with genera such as Grevilleawhich appear to be nested well within the family (Hootand Douglas, 1998) and exhibit highly derived endo-sperm developmental patterns (Venkata Rao, 1967). Allmembers of the Proteaceae exhibit free nuclear endo-sperm development (Johri, Ambegaokar, and Srivastava,1992). While derived taxa have been studied extensively,there is a single report of endosperm development in thebasal genus Bellendena (Hoot and Douglas, 1998). Thistaxon shares many embryological features with Platanus:an orthotropous ovule, micropyle formed by the innerintegument only, distinct chalazal and micropylar zonesin the free nuclear endosperm, and a large embryo in themature seed (Venkata Rao, 1967). However, Bellendenareportedly has micropylar-to-chalazal initiation of endo-sperm cellularization (Venkata Rao, 1967), the oppositeof Platanus. The similarities between Platanus and Bel-lendena are consistent with the hypothesis that they sharea single evolutionary origin of free nuclear endosperm ina common ancestor. A more complete analysis of endo-sperm development in basal Proteaceae and Nelumbo isneeded to provide a broader basis for comparison withinthis intriguing clade. It is also clear that comparative em-bryology has great potential to address questions of re-lationship in the Platanaceae/Proteaceae/Nelumbonaceaeclade, which is otherwise difficult given the highly spe-cialized and apomorphic nature of the sporophyte phasein these three families.

Few developmental analyses of endosperm are avail-able for other basal eudicot taxa. However, several recentreports describe aspects of endosperm development fortwo taxa in the other two putative ‘‘free nuclear’’ basaleudicot clades (Fig. 46): Ranunculus (Ranunculaceae)(Chitralekha and Bhandari, 1993; XuHan and Van Lam-


Fig. 47. Phylogenetic relationships of the families with free nuclearendosperm compared in the text. Each group represents a monophyleticlineage. Tree based on Chase et al. (1993).

meren, 1993, 1997) and Papaver (Papaveraceae) (Olson,1981). It should be noted that neither Ranunculus norPapaver are basal genera within Ranunculaceae and Pa-paveraceae (Hoot et al., 1997; Ro, Keener and McPheron,1997). Our comparison is thus limited to taxa nestedwithin the two lineages and thus with potentially morederived characteristics. Fortunately, there do not appearto be widely deviant patterns of endosperm developmentwithin these two families as has been observed in theProteaceae (see discussion above) (Johri, Ambegaokar,and Srivastava, 1992, and references therein). In addition,the Menispermaceae represent a more basal branch in theranunculid free nuclear clade than the Ranunculaceae(Fig. 46), yet embryological reports for taxa in the Men-ispermaceae (Sastri, 1964), although somewhat lackingin detail, appear to show congruence with descriptionsfor the Ranunculaceae. Thus, the use of Papaver and Ra-nunculus as representatives of the two putative free nu-clear 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 avail-able) from Platanus and Ranunculus (Chitralekha andBhandari, 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 fila-mentous embryo stage (Olson, 1981) as does Platanus,but differs from Platanus in some other embryologicalfeatures including having a micropyle formed by bothinteguments and a small embryo surrounded by abundantendosperm in the mature seed (Johri, Ambegaokar, andSrivastava, 1992).

Ranunculus shares some features of endosperm devel-opment with Platanus including chalazal-to-micropylarpolarity of anticlinal cell wall initiation, wall initiationassociated with mitosis, and the storage of oil and protein.However, Ranunculus also differs from Platanus in theduration of cellularization, embryo developmental stageat endosperm anticlinal wall initiation, and mode of cen-tripetal wall formation. In Platanus, some anticlinal wallsin the micropylar zone appear to converge as they growin toward the central vacuole, closing some of the alveoliformed when anticlinal walls are initiated (Fig. 42). Cen-tripetal cellularization continues to form cells of varyingsize and with no distinct layering (‘‘irregular’’ in Table1) (Fig. 43). In contrast, anticlinal walls in Ranunculusgrow inward without converging (Chitralekha and Bhan-dari, 1993). Periclinal walls form after rounds of mitosisso that regular layers of cells are produced centripetally(‘‘regular’’ in Table 1) (Chitralekha and Bhandari, 1993).In addition to the characters compared in Table 1, Pla-tanus embryos are large and well developed at seed ma-turity and are surrounded by a thin layer of endosperm.In Ranunculus, embryos only reach an early cotyledonstage and are surrounded by copious amounts of endo-sperm at seed maturity (XuHan and Van Lammeren,1997). Clearly there is variability among these three basaleudicot taxa (Platanus, Ranunculus, and Papaver) (Fig.46).

An even greater degree of developmental variability isevident among free nuclear endosperms when compari-sons are made with higher eudicot taxa, represented hereby legumes (Fabaceae) (Yeung and Cavey, 1988; Dute

and Peterson, 1992; Chamberlin, Horner, and Palmer,1994; XuHan and Van Lammeren, 1994; Algan and Bak-ar, 1996), Stellaria (Caryophyllaceae) (Newcomb andFowke, 1973), and Helianthus (Asteraceae) (Newcomb,1973) (Fig. 47). These three lineages all have two similarfeatures: micropylar-to-chalazal anticlinal wall initiationand irregular centripetal cellularization (Table 1). In gen-eral, these higher eudicot taxa have less extensive freenuclear development prior to cellularization than the freenuclear basal eudicots, although there is variability in thisfeature. Cellularization begins when fewer than ten freenuclei are present in Helianthus (Newcomb, 1973),whereas many free nuclei are produced before walls formin Stellaria (Newcomb and Fowke, 1973) and legumes(Algan and Bakar, 1996). Endosperm cell wall initiationin the higher eudicots appears to occur at a relativelyadvanced embryo stage, but this feature also variesamong the higher eudicots. The embryo reaches theheart-shaped stage before walls are initiated in Stellaria(Newcomb and Fowke, 1973), but wall formation beginsat the globular embryo stage in legumes (Yeung and Cav-ey, 1988; Dute and Peterson, 1992; Chamberlin, Horner,and Palmer, 1994) and Helianthus (Newcomb, 1973). Ineach of these cases, the onset of endosperm cellulariza-tion occurs at a much more advanced embryo stage thanin Platanus where the embryo is in an early filamentousphase when walls begin to form in the chalazal zone (Ta-ble 1).

Both legumes (Chamberlin, Horner, and Palmer, 1994;Algan and Bakar, 1996) and Helianthus (Newcomb,1973) store protein and oil, like Platanus, Ranunculus,and Papaver, although in some legumes the endospermdoes not acquire any storage compounds (Chamberlin,


Horner, and Palmer, 1994). There has been no report ofendosperm storage products in Stellaria. Endosperm de-velopment patterns vary greatly among this sample ofhigher eudicots (legumes, Helianthus, Stellaria); and theyall differ in many ways from basal eudicots (Table 1).

Finally, we can extend the comparison of free nuclearendosperms to include the group most distantly relatedto Platanus, i.e., the monocots (Figs. 46, 47). The basalmonocot Acorus (Chase et al., 1993; Duvall et al., 1993a,b; Nandi, Chase, and Endress, 1998) is reported to haveab initio cellular endosperm (Buell, 1938). This, alongwith parsimonious interpretation of character distribution(Fig. 46), indicates that at some point during the separateradiations of monocot and eudicot clades from magnoliidancestors, free nuclear endosperms were also derived in-dependently from the ab initio cellular pattern of devel-opment in the monocot clade.

Cereal endosperm development has been well studiedand characterized (Mares, Norstog, and Stone, 1975;Morrison and O’Brien, 1976; Mares et al., 1977; Fineran,Wild, and Ingerfeld, 1981; Van Lammeren, 1988; Engell,1989; Bosnes, Weideman, and Olsen, 1992; Olsen, Potter,and Kalla, 1992; Brown, Lemmon, and Olsen, 1994,1996; Olsen, Brown, and Lemmon, 1995) and exhibits aunique combination of features (Table 1). Extensive freenuclear development occurs, as in Platanus, but cellular-ization is completed rapidly after 1 wk rather than 2 mo(Olsen, Brown, and Lemmon, 1995). Cereals are the onlygroup of the eight compared in Table 1 to store abundantstarch in the endosperm. Another unusual feature of ce-real endosperm is a regular centripetal cellularization,which is shared only with Ranunculus (XuHan and VanLammeren, 1993) among the eight taxa compared. How-ever, unlike Ranunculus, cereal endosperms exhibit dif-ferentiation into aleurone and starchy layers that storehydrolytic protein and starch, respectively (Olsen, Potter,and Kalla, 1992; Olsen, Brown, and Lemmon, 1995).This pattern is unique among free nuclear taxa. There aresome similarities in the endosperm of cereals and severalother free nuclear taxa, such as micropylar-to-chalazalcellularization (Johri, Ambegaokar, and Srivastava, 1992)(present in all taxa except Platanus and Ranunculus) andwall initiation at the globular proembryo phase (sharedwith legumes and Helianthus) (reviewed in Olsen,Brown, and Lemmon, 1995).

Comparison of free nuclear endosperm developmentamong taxa also reveals that Platanus endosperm exhibitssome features that are unique or at least unusual. Theseinclude a distinct chalazal zone that cellularizes first, dif-ferential cellularization patterns of the chalazal and mi-cropylar zones, a multinucleate cellular stage in the cha-lazal zone, chalazal-to-micropylar cellularization, and dif-ferentiation into an inner endosperm that is consumed bythe embryo and a persistent outer endosperm that is filledwith storage products.

When features of development are compared, it is clearthat free nuclear endosperms are not all the same. Thereare differences in almost every aspect of developmentaltiming, patterning, and structure among the three basaleudicot lineages with free nuclear endosperm (represent-ed by Platanus, Papaver, and Ranunculus). An evengreater degree of variability is evident when comparisonswith Platanus are extended to include more distantly re-

lated, free nuclear taxa in the higher eudicots (representedby legumes, Stellaria, and Helianthus) and monocots(represented by cereals) (Figs. 46, 47; Table 1). Thesefindings are congruent with the hypothesis that free nu-clear endosperm has evolved independently within basaleudicots, higher eudicots, and monocots.

The cellular-to-free nuclear transition: developmentalperspective—An intriguing feature observed in Platanuswas the presence of an incipient cell plate betweendaughter nuclei derived from the primary endosperm nu-cleus (Fig. 32). Although interzonal phragmoplasts thatfail to form cell plates have been observed during freenuclear mitosis of cereal endosperm (Olsen, Brown, andLemmon, 1995), this is the first report of this kind ofphenomenon in a more basal angiosperm. Both of thesestructures are suggestive of an ab initio cellular ancestryfor free nuclear endosperm, and this is in turn consistentwith the hypothesis that ab initio cellular development isplesiomorphic for angiosperms (Donoghue and Scheiner,1992; Friedman, 1992). The evolutionary transition to afree nuclear condition would have involved a develop-mental disruption of normal cytokinesis. The point of dis-ruption appears to be different in Platanus and cereals:in Platanus cytokinesis is interrupted at a point after cellplate initiation but before cell wall completion; in cerealscytokinesis is interrupted after phragmoplast formationbut before a cell plate is formed. This again is consistentwith the hypothesis of independent origins of free nuclearendosperm in these two groups. It would be interestingto know whether vestiges of cytokinesis are evident intaxa, other than Platanus and cereals, with free nuclearendosperm.

Conclusions—Our analysis of embryology in Platanusreveals much more complexity than traditional typologi-cal designations suggest, particularly for endosperm. Bycarefully examining the entire developmental process, wehave observed a number of endosperm features that havenot been explicitly reported in basal taxa before, such asa transitory cell plate during free nuclear mitosis, chalazaland micropylar zones with distinct modes of cellulariza-tion, and unique patterns of cellular differentiation.

The initial stage of endosperm development in Platan-us (free nuclear) is completely different from what waspreviously reported (ab initio cellular). Two other basalangiosperm taxa, Drimys winteri (Johri, Ambegaokar, andSrivastava, 1992) and Lactoris (Tobe et al., 1993), haverecently been shown to be ab initio cellular in contrast toearlier reports of free nuclear development. This dem-onstrates the critical need for modern reevaluation of em-bryological characters for key basal angiosperm taxa. In-correct data will lead to erroneous conclusions aboutcharacter evolution and relationship.

Comparison of embryology in Platanus with limitedpublished data for basal Proteaceae indicates possibleevolutionary homologies, congruent with the hypothesisof recent shared ancestry and a common origin of freenuclear endosperm development in the clade includingPlatanaceae and Proteaceae. In contrast, comparison ofendosperm developmental characters in Platanus withother free nuclear taxa reveals many significant differ-ences. This is consistent with the hypothesis that free


nuclear endosperms have evolved numerous times withinangiosperms, including three times during the early ra-diation of the eudicot clade.

Comparative analysis of endosperm development with-in the appropriate phylogenetic context has the potentialto yield insight into reproductive character evolution, par-ticularly at the base of the eudicot clade where there isconsiderable variability. However, analyses with the nec-essary level of detail are mostly restricted to phyloge-netically derived groups such as cereals and legumes thatare distantly related to each other and to Platanus. With-out comparable data sets for other basal taxa, a full ex-ploration of the evolutionary history of the features wehave reported is not possible. Future embryological in-vestigations must include additional basal eudicots, basalmonocots, and magnoliids in order to permit broad, com-parative analyses of these defining angiosperm reproduc-tive characters. The results are likely to reveal importantnew insights into the early evolutionary radiation of flow-ering plants.

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