Plant Syst. Evol. 228: 153±169)2001)

Developmental evolution of endosperm in basal angiosperms: evidence from Amborella Amborellaceae), Nuphar Nymphaeaceae), and Illicium Illiciaceae)

S. K. Floyd and W. E. Friedman

Department of Environmental, Population, and Organismic Biology, University of Colorado, Boulder, Colorado, USA

Received January 19, 2001 Accepted March 19, 2001

Abstract. Because of their basal phylogenetic posi- Within the last two years remarkable progress tion, Amborella, Nymphaeales, and Illiciales )and has been made toward the resolution of deep allies) are key to reconstructing ancestral character angiosperm phylogenetic relationships. The states and to tracing character state transitions that results of several independent, molecular se- occurred during the earliest radiation of ¯owering quence-based analyses have converged on plants. Endosperm is the sexually-derived embryo- similar results for rooting the angiosperm tree nourishing tissue that is unique to the life cycle of and have identi®ed the three earliest-diverging angiosperms. We provide detailed descriptions of endosperm development in Amborella, Nuphar lineages of ¯owering plants )Mathews and )Nymphaeales), and Illicium )Illiciales) and com- Donoghue 1999; Parkinson et al. 1999; Qiu pare patterns within an explicit phylogenetic con- et al. 1999, 2000; Soltis et al. 1999, 2000; text for the three basal lineages that they represent. Borsch et al. 2000; Graham and Olmstead Amborella and Illicium share a bipolar, cellular 2000; Savolainen et al. 2000). The consensus of pattern of development, characterized by an these analyses is that Amborella trichopoda is oblique ®rst division, that was resolved as ancestral sistertoallotherextantangiosperms,Nymphae- for ¯owering plants. A series of character state ales )Nymphaeaceae plus Cabombaceae) is the transformations occurred within Nymphaeales sister group to all taxa except Amborella, and a which led ®rst to a modi®ed cellular pattern with clade including Illiciales, Austrobaileyaceae, a transverse ®rst division )present in Nuphar). The and Trimeniaceae )hereafter referred to as the transverse cellular pattern was transformed to a Illiciales clade) is sister to all remaining helobial pattern that is present in Cabombaceae. Endosperm ontogeny involves dissociable elements angiosperms )Fig. 1). With identi®cation of and appears to ®t the model of a modular devel- the three, earliest-diverging lineages of ¯ower- opmental process. ing plants )the ``basal grade''), it is now possible to more reliably reconstruct ancestral Key words: Amborella, angiosperm embryology, character states for the angiosperm clade development, endosperm, evolution, EvoDevo, )Mathews and Donoghue 1999, Soltis et al. helobial, Illicium, modularity, Nuphar. 1999, Friedman and Floyd 2001). 154 S. K. Floyd and W. E. Friedman: Developmental evolution of endosperm

Recent comparative investigations of en- dosperm in a broad sample of basal ¯owering plants )Floyd et al. 1999, Floyd and Friedman 2000) revealed new insights into the nature of this unique component of angiosperm repro- ductive biology that go beyond traditional typology. These analyses demonstrate that endosperms of most ancient angiosperm lin- eages exhibit a cellular ontogeny that is resolved as ancestral, based on phylogenetic comparative analysis. This pattern is charac- terized by an unequal division of the ®rst endosperm cell, producing a small chalazal cell and larger micropylar cell. Early development of the micropylar region )transverse cell divi- sions) results in a few large cells in a uniseriate arrangement. Early development of the chala- zal region involves cell divisions in many Fig. 1. Consensus of several recent molecular se- planes. Characterization of a primitive ontog- quence based phylogenies for angiosperms )Mathews eny for endosperm provides the basis for and Donoghue 1999; Qiu et al. 1999, 2000; Soltis tracing the evolution of endosperm within the et al. 1999, 2000; Barkman et al. 2000; Graham and ¯owering plant clade. Olmstead 2000) In addition to proposing an ancestral ontogeny for endosperm, Floyd and Friedman Reproductive characters constitute the ma- )2000) have shown that three features of early jority of the unique features that separate endosperm development de®ne the basic pat- angiosperms from all other seed plants )Sar- tern. These are division of the primary endo- gant 1908, Crane et al. 1995, Friedman 2001). sperm nucleus or cell, development of the Thus, knowledge of the reproductive biology chalazal domain, and development of the of basal lineages of ¯owering plants is critical micropylar domain. Analyzed as characters to the reconstruction of ancestral character with variable character states, these three states and key to understanding the origin and features appear to have evolved independently early history of the angiosperm clade. One of within angiosperms, resulting in variable these unique angiosperm reproductive features endosperm patterns. is endosperm, the embryo-nourishing tissue Although many basal angiosperms have that develops following a fertilization event retained the primitive cellular ontogeny de- involving a second sperm and the two haploid scribed above, endosperm ontogenetic evolu- polar nuclei of the female gametophyte. tion has occurred in some lineages so that all Traditionally, endosperm has been classi- three endosperm types are represented among ®ed into three types based on di€erential early-divergent angiosperms. In particular, patterns of development: ``free nuclear,'' in helobial development occurs in Cabomba,a which early mitotic divisions occur without member of Nymphaeales )Floyd and Fried- cytokinesis; ``ab initio cellular'' )cellular), in man 2000), one of the three lineages of the which cell walls are formed following all basal grade. The explicit phylogenetic hypoth- mitotic divisions; and ``helobial,'' involving esis for the branching order of Amborella, an initial transverse cellular division followed Nymphaeales and Illiciales, and recent resolu- by free nuclear development of the micropylar tion of relationships within Nymphaeales )Les cell or chamber. et al. 1999), provide the context in which to S. K. Floyd and W. E. Friedman: Developmental evolution of endosperm 155 reconstruct endosperm character polarity and Materials and methods evolution in general, and more speci®cally to Amborella trichopoda was collected in New Cale- explore the origin of helobial endosperm in donia and chemically ®xed in either FAA or 4% Nymphaeales. glutaraldehyde in Sorensen's bu€er )pH 6.8; Very few detailed analyses of endosperm Electron Microscopy Sciences). Illicium ¯oridanum development have been published for taxa in and Nuphar lutea ssp. polysepala were collected in the basal grade. Tobe et al. )2000) described Georgia and Colorado, respectively, and brought some basic aspects of endosperm development to the laboratory in Boulder, Colorado, where for Amborella within the context of a broader specimens were chemically ®xed with 4% acrolein embryological study. Floyd and Friedman in 50 mM Pipes bu€er )also 5 mM EGTA and )2000) also reported that Amborella and 1 mM MgSO4) at pH 6.8. Specimens of Illicium Illicium exhibit bipolar, cellular endosperm mexicanum were shipped overnight from the Uni- development. Limited data were previously versity of California Botanical Garden, Berkeley, and chemically ®xed with acrolein as described available for the Illiciales clade )Hayashi above. Collections are summarized in Table 1. 1963a, b). Nymphaeales have been the subject More than 865 female ¯owers and developing of several embryological studies )Cook 1902, seeds were serially sectioned for this analysis 1906, 1909; Khanna 1965, 1967; Ramji and )Table 1). The presence of proteins, lipids, and Padmanabhan 1965; Padmanabhan 1970; starch in mature endosperm tissue was determined Schneider 1978; Galati 1985; Van Miegroet with histochemical stains and cross polarization and Dujardin 1992; Orban and Bouharmont microscopy. Histological methods followed Floyd 1995), but remarkably, complete descriptions and Friedman )2000). Endosperm characters were of endosperm development are lacking for all parsimoniously optimized onto published clado- taxa except Cabomba )Floyd and Friedman grams using MacClade )Maddison and Maddison 2000) and Nymphaea )Cook 1906). Endo- 1992). sperm development in Nuphar, which has been resolved as sister to all other Nymphae- Results aceae )Les et al. 1999), has never been de- scribed. Amborella. Endosperm development in Ambo- We report here for the ®rst time detailed rella begins with migration of the primary descriptions of endosperm development in endosperm nucleus to the extreme chalazal end Amborella, Nuphar, and Illicium. Endosperm of the large ®rst endosperm cell )central cell) ontogenetic patterns are then compared within where it undergoes mitosis )Fig. 2A, B). Fol- the framework of recent cladistic analyses for lowing the initial mitotic division, an oblique basal angiosperms. Based on comparative cell wall is formed that unequally partitions the analysis, we elaborate on the hypothesis for ®rst endosperm cell into a small chalazal cell the ancestral bipolar, cellular endosperm pat- and a much larger micropylar cell )Fig. 2C). tern outlined by Floyd and Friedman )2000). The larger micropylar cell normally under- A scenario is then presented for endosperm goes one or two highly unequal transverse/ ontogenetic evolution among the three basal oblique cell divisions at its chalazal end angiosperm lineages )basal grade) that pro- )Fig. 2D) until a single, large cell at the vides the ®rst clear hypothesis for the deriva- micropylar pole is de®ned by a roughly trans- tion of a helobial pattern from ab initio cellular verse wall that crosses the full width of the endosperm based on the interpretation of former ®rst endosperm cell )Fig. 2E±H). The character distribution within an explicit, phy- chalazal cell, derived from the ®rst cell division logenetic context. We then brie¯y discuss the of the endosperm, also divides, most often in a relevance of the concept of modularity in vertical plane )Fig. 2F). development to our hypothesis of endosperm The derivatives of the chalazal cell along ontogenetic evolution. with the chalazal derivatives of the micropylar 156 S. K. Floyd and W. E. Friedman: Developmental evolution of endosperm

Table 1. Source and voucher collection information for ¯oral and fruit materials used in this study Taxa Source Herbarium voucher or Number garden accession number specimens sectioned Amborella trichopoda Baillon Leonard Thien, Tulane #1000 in liquid >450 University specimen Mt. Aoupinie , New COLO Floyd and Caledonia Lemieux 00-24 Nuphar lutea Sibthorp & Smith Red Rocks Lake, Floyd and Williams 00-36- >85 ssp. polysepala )Engelmann) Colorado 00-49in liquid specimen, Beal Boulder, Colorado Illicium ¯oridanum Ellis. Cultivated, Georgia 95-0278 >178 State Botanical Garden, Athens GA Illicium mexicanum A. C. Smith Cultivated, University 91.0030 >157 of California Botanic Garden, Berkeley, CA cell )of the two-celled endosperm) then under- regions that are the result of di€erential go repeated cell divisions in many planes developmental rates and patterns. One of resulting in a mass of cells at the chalazal these is a region at the chalazal end of the end of the endosperm )Fig. 2G, H). During endosperm that is multicellular and multiseri- this period of active cellular development at ate and comprises relatively small and cyto- the chalazal end of the endosperm, the large plasmically dense cells. We refer to this micropylar cell remains undivided or divides region as the chalazal domain )Fig. 2G, H). once by means of a transverse wall )Fig. 2H). The micropylar domain of the endosperm Thus early stages of ontogeny produce two comprises either a single large vacuolate cell

c Fig. 2. Endosperm development in Amborella. All sections longitudinal with micropylar end at the top of the page. A Primary endosperm nucleus )pen) at chalazal end of central cell )cc). Scale bar ˆ 25 lm. B Primary endosperm nucleus in metaphase )arrow indicates plate); inset shows mitotic ®gure at higher magni®cation, Scale bar ˆ 25 lm. C Two-celled endosperm, with smaller chalazal cell )ch), oblique partitioning cell wall )arrow), and large micropylar cell )mc). D Nucleus of the micropylar cell in mitosis )half of anaphase ®gure in view), chalazal cell )ch) undivided, original oblique wall indicated by arrow; inset shows mitotic ®gure at higher magni®cation. Scale bar ˆ 25 lm. E Three-celled endosperm. A cell wall has formed following mitosis of the micropylar cell; chalazal cell )ch) remains undivided, original oblique wall indicated by arrow. Zygote )z) remains undivided. Scale bar ˆ 25 lm. F Four-celled endosperm comprising two derivatives from each of the ®rst two cells. Endosperm is now di€erentiated into a large, unicellular micropylar domain )md) and smaller, multicellular )three cells) chalazal domain )cd). Scale bar ˆ 25 lm. G Cell division in many planes in the chalazal domain )cd) have produced seven cells in a multiseriate arrangement; micropylar domain )md)remains unicellular and zygote )z) remains undivided. Scale bar ˆ 25 lm. H Most advanced di€erentiated endosperm observed; chalazal domain composed of 12 cells, with those at extreme chalazal end small and densely cytoplasmic. I±K Partitioning of micropylar domain into several large, vacuolate cells in a multiseriate arrangement; zygote )z) remains undivided. Scale bars ˆ 50 lm. L Copious, multicellular endosperm )end)and embryo )emb) of mature seed )seed coat removed). Scale bar ˆ 500 lm. M Higher magni®cation view of mature endosperm cells showing numerous, angular protein bodies. Scale bar ˆ 50 lm S. K. Floyd and W. E. Friedman: Developmental evolution of endosperm 157 158 S. K. Floyd and W. E. Friedman: Developmental evolution of endosperm or two large cells in a uniseriate arrangement The chalazal domain remains unicellular )Fig. 2D). and uninucleate and continues to grow as an Following this highly di€erentiated early invasive tube )haustorium) to the extreme phase, the micropylar domain becomes devel- chalazal end of the massive nucellus opmentally active. The cell or cells of the )Fig. 3C±G). The nucleus of the chalazal micropylar domain divide, resulting in a mul- domain cell ultimately migrates to the chalazal tiseriate arrangement )Fig. 2I±K). Continued end )Fig. 3F). The ®rst cell of the micropylar growth and cell division occur in both domains domain divides once transversely to yield two to produce a mature endosperm that consists of cells in an uniseriate arrangement )Fig. 3D, E). small, uniform cells that are ®lled with lipids and Cell divisions in many planes then occur in protein bodies )Fig. 2L, M). Starch is absent. the micropylar domain leading to a multicel- The mature seed contains copious endosperm lular, multiseriate arrangement that initially and a rudimentary embryo )Fig. 2L). retains the narrow, cylindrical shape of the Nuphar. The primary endosperm nucleus micropylar cell )Fig. 3F, G). Lateral expansion divides in a position slightly nearer to the of the micropylar domain then occurs, associ- micropylar end than to the chalazal end of the ated with continued cell divisions. The embryo elongate central cell )Fig. 3A). Following divi- grows into the micropylar endosperm and sion, a transverse wall is formed that divides the consumes all but a thin layer of cellular tissue central cell unequally into a large chalazal cell that remains in the mature seed )Fig. 3H±K). and a smaller micropylar cell )Fig. 3B, C). Cells of the mature micropylar endosperm These two cells precisely de®ne the two regions contain lipids and protein bodies. The chalazal that will undergo di€erential development, the domain is still present in the mature seed, but chalazal and micropylar domains. its long tubular shape renders it dicult to see

c Fig. 3. Endosperm development in Nuphar. All sections longitudinal with micropylar end at the top of the page. A Primary endosperm nucleus )arrow) in mitosis )anaphase), zygote )z) undivided. Scale bar ˆ 25 lm. B Developing seed at the two-celled endosperm stage, with larger chalazal cell )ch), transverse partitioning cell wall)arrow),andsmallmicropylarcell)mc); zygote )z) undivided. The nucleus of the chalazal cell has migrated toward the chalazal end. Narrow, tubular endosperm surrounded by massive nucellus tissue which will develop into perisperm )p). Scale bar ˆ 50 lm. C Higher magni®cation of the two-celled endosperm in 3B, scale bar ˆ 50 lm. D Developing seed at the three-celled endosperm stage, composed of two derivatives of the micropylar cell and the undivided chalazal cell. Both nuclei of the two micropylar derivatives are in view. Arrow indicates ®rst transverse wall. Endosperm is now di€erentiated into a uniseriate )two cells) micropylar domain )md) and unicellular chalazal domain )cd). Narrow, tubular endosperm surrounded by massive nucellus tissue which will develop into perisperm )p). Scale bar ˆ 50 lm. E Higher magni®cation of the two-celled endosperm in 3D, scale bar ˆ 50 lm. F Developing seed at a later di€erentiated stage; seed now about twice the size as in 3D. The chalazal domain )cd) remains unicellular and has grown closer to the chalazal end of the perisperm )p); its nucleus has enlarged and migrated to the chalazal end of the tubular cell. The micropylar domain )md)is multicellular and multiseriate. The inset shows di€erent section through the same endosperm to show the nucleus of the chalazal domain at the extreme chalazal end. Scale bar ˆ 100 lm. G Higher magni®cation of the two-celled endosperm in 3F; arrow indicates position of ®rst transverse wall. Scale bar ˆ 100 lm. H Micropylar domain has expand laterally, forming a multicellular region of endosperm )end) into which the developing embryo )emb) has begun to grow. Scale bar ˆ 250 lm. I Slightly later stage than 3H, embryo )emb)has consumed much of the micropylar endosperm )end). Scale bar ˆ 250 lm.JMatureseedwiththinlayerof multicellular micropylar endosperm surrounding the well-developed embryo )emb) occupying the micropylar end of the seed, adjacent to abundant perisperm tissue )p). Chalazal endosperm not in view. Scale bar ˆ 500 lm. K Higher magni®cation view of embryo )emb) and micropylar endosperm )arrows) in the mature seed shown in 3J. Scale bar ˆ 500 lm S. K. Floyd and W. E. Friedman: Developmental evolution of endosperm 159 except in serial sections. Perisperm occupies nucleus from its initial position in the ®rst most of the mature seed )Fig. 3J). The peri- endosperm cell )midway between chalazal and sperm cells are ®lled with packets of starch micropylar poles) to the extreme chalazal end grains. )Fig. 4A). Following mitosis of the primary Illicium. Endosperm development begins endosperm nucleus, an oblique cell wall is with migration of the primary endosperm formed that unequally partitions the ®rst 160 S. K. Floyd and W. E. Friedman: Developmental evolution of endosperm endosperm cell into a small chalazal cell and a The entire endosperm then undergoes cellular much larger micropylar cell )Fig. 4B). Both the division and growth )Fig. 4H, I). Ultimately chalazal cell and the micropylar cell undergo the entire endosperm consists of small, uniform further divisions )Fig. 4C±F). cells that are ®lled with lipids and protein Beginning at the chalazal end, the large bodies )Fig. 4I, J). Starch is absent. The mature micropylar cell undergoes a few transverse cell seed contains abundant endosperm and a divisions that result in one to a few small cells rudimentary embryo )Fig. 4I). adjacent to the chalazal cell, and a few larger cells in a uniseriate arrangement at the micro- Discussion pylar end of the endosperm )Fig. 4D, E). The results of these ®rst few divisions are the Comparison of endosperm developmental pat- formation of the micropylar and chalazal terns in the basal angiosperm grade. Both domains that will undergo di€erential devel- Amborella and Illicium endosperms exhibit all opment. of the features of the bipolar, cellular devel- The micropylar domain, comprising the opmental pattern that was hypothesized as uniseriate derivatives of the micropylar cell, ancestral for angiosperms by Floyd and Fried- continues limited uniseriate development man )2000). The early pattern-forming phase )Fig. 4D, F). The chalazal domain includes of this primitive ontogeny involves unequal, derivatives of both the original chalazal cell cellular partitioning of the ®rst endosperm cell and the micropylar cell )of the two-celled into a large micropylar cell and a small endosperm) that both divide in several planes chalazal cell. The initial division takes place giving rise to a multiseriate mass of cells at the chalazal end of the ®rst endosperm cell, )Fig. 4C±F). Continued cellular development following the migration of the primary endo- results in a mass of up to 35 cells in the chalazal sperm nucleus to that position. Following the domain, while the micropylar domain remains initial division, limited cellular uniseriate few-celled and uniseriate )Fig. 4F). Following development occurs in the micropylar domain the early phase of di€erential development, the )usually just one or no divisions in Amborella), micropylar domain becomes multiseriate in a and more extensive cellular, multiseriate de- chalazal-to-micropylar direction )Fig. 4G). velopment takes place in the chalazal domain.

c Fig. 4. Endosperm development in Illicium. All sections longitudinal with micropylar end at the top of the page. A Primary endosperm nucleus )pen)atchalazalendofcentralcell)cc). Zygote )z) remains undivided. Scale bar ˆ 25 lm. B Two-celled endosperm, with smaller chalazal cell )ch), oblique partitioning cell wall )arrow), and large micropylar cell )mc). Zygote )z) remains undivided. Scale bar ˆ 25 lm. C Three-celled endosperm. Micropylar cell )mc) undivided; chalazal cell has divided into two cells which now constitute the chalazal domain )cd). Scale bar ˆ 25 lm. D Four-celled endosperm comprised of three derivatives of the micropylar cell and the chalazal cell )still undivided). Endosperm is now di€erentiated into a large, uniseriate )two cells) micropylar domain )md) and smaller, multicellular )two cells) chalazal domain )cd). The original oblique wall is indicated by the arrow. The zygote )z) remains undivided. Scale bar ˆ 25 lm. E Later di€erentiated stage. The chalazal domain )cd) comprises several cells in a multiseriate arrangement; the micropylar domain )md) comprises 3 cells in a uniseriate arrangement. The zygote )z) remains undivided. Scale bar ˆ 25 lm. F Most advanced di€erentiated stage. The chalazal domain )cd) comprises about 35 cells; the micropylar domain )md) comprises 4 cells. Scale bar ˆ 50 lm. G Endosperm )end) after elongation and continued cell division. Micropylar domain partially multiseriate. The zygote )z) remains undivided. Scale bar ˆ 50 lm. H Endosperm )end) completely multiseriate and beginning to expand in girth at expense of massive nucellus )n). Scale bar ˆ 300 lm. I Copious, multicellular endosperm )end)andembryo)emb)ofmature seed )seed coat mostly removed). Scale bar ˆ 500 lm. J Higher magni®cation of mature endosperm cells showing numerous protein bodies, stained blue, surrounded by unstained lipids. Scale bar ˆ 50 lm S. K. Floyd and W. E. Friedman: Developmental evolution of endosperm 161

Amborella and Illicium also share some addi- the two domains that are subsequently de®ned. tional aspects of early endosperm patterning. An essentially identical pattern of development In both taxa, division of the ®rst endosperm was observed in Drimys winteri )Floyd and cell occurs by formation of an obliquely- Friedman 2000), and may also occur in positioned wall, the chalazal domain is formed another member of Winteraceae, Pseudowin- from derivatives of both of the ®rst two tera colorata )Bhandari 1963). Thus the endo- endosperm cells, and the micropylar domain sperm developmental pattern present in initially comprises a single derivative of the Amborella and Illicium also occurs in at least micropylar cell of the two-celled endosperm one later-diverging angiosperm group )Fig. 1). )Figs. 2, 4). In other words, the ®rst endo- The presence of a pattern of endosperm sperm cell division corresponds imprecisely to development characterized by an oblique ®rst 162 S. K. Floyd and W. E. Friedman: Developmental evolution of endosperm division )that imprecisely de®nes micropylar and chalazal domains) in two of the three lineages of the basal grade )Amborella and the Illiciales clade) suggests that this may represent a shared ancestral condition. In contrast, early endosperm development in Nuphar involves a transverse ®rst division into two cells that directly correspond to the micropylar and chalazal domains )Fig. 3). As in Amborella and Illicium, the ®rst endosperm wall forms at the position of the primary endosperm nucleus. But in Nuphar, the primary endosperm nucleus does not migrate to the chalazal end, hence the ®rst wall is closer to the micropylar end than in Amborella and Illicium. The chalazal Fig. 5. Diagrammatic comparison of three patterns of endosperm development that occur in lineages of domain develops as a unicellular, uninucleate the basal angiosperm grade. Darker gray shading tubular structure in Nuphar, in contrast to indicates the part of the endosperm derived from the cellular multiseriate chalazal development in chalazal cell, lighter gray shading highlights micro- both Amborella and Illicium. Micropylar devel- pylar cell development. A The cellular endosperm opment is cellular, with an initial uniseriate developmental pattern that occurs in Amborella and phase in all three taxa described above. Illicium. The ®rst division is oblique, the micropylar The same basic pattern of development domain )md) is derived exclusively from a derivative observed in Nuphar seems to occur in most of the micropylar cell and the chalazal domain )cd)is other members of Nymphaeaceae, including derived from both the chalazal and micropylar cells. Nymphaea, Castalia )Cook 1906), and Victoria B The cellular endosperm developmental pattern that )Khanna 1967). Free nuclear development has occurs in Nuphar )Nymphaeaceae). The ®rst division been reported to occur in Euryale )Khanna is transverse, the micropylar domain )md)isderived from the micropylar cell and the chalazal domain )cd) 1964), but additional studies are needed to is derived the chalazal cell. C The helobial endosperm con®rm this ®nding. Both genera of Cabomb- developmental pattern that occurs in Cabomba aceae, sister group to Nymphaeaceae )Les )Cabombaceae). The ®rst division is transverse, the et al. 1999), exhibit patterns of endosperm micropylar domain )md) is derived from the micro- development that are very similar to the pylar cell and the chalazal domain )cd) is derived the cellular pattern in Nymphaeaceae with one chalazal cell. Early development of the micropylar notable di€erence. In both Cabomba and domain is free nuclear Brasenia, micropylar development begins with a free nuclear stage, followed by centripetal lular ontogenies are initiated by an unequal cellularization )Cook 1906, Floyd and Fried- cellular division, followed by cellular, uniseri- man 2000), which constitutes a helobial pat- ate development of the micropylar domain. tern of development. The transverse/cellular pattern of Nuphar is Three endosperm developmental patterns also comparable to the transverse/helobial may therefore be recognized among taxa in the pattern of Cabomba in that both are charac- basal angiosperm grade )Fig. 5): oblique/cel- terized by an initial unequal, transverse cell lular )Amborella and Illicium) )Fig. 5A), trans- division and unicellular development of the verse/cellular )Nuphar) )Fig. 5B), and chalazal domain. If we accept the hypothesis transverse/helobial )Cabomba) )Fig. 5C). The that the oblique/cellular pattern is ancestral, transverse/cellular pattern of Nuphar bears comparison of patterns suggests a transforma- developmental similarities to both of the other tional series from the oblique/cellular pattern, patterns. Transverse/cellular and oblique/cel- through a transverse/cellular intermediate, to a S. K. Floyd and W. E. Friedman: Developmental evolution of endosperm 163 derived transverse/helobial pattern )Fig. 5A± Illiciales clade, and the most recent common C) in Nymphaeales. However, to make a ancestor of all remaining angiosperms. A strong statement about ontogenetic evolution, transformation to transverse/cellular occurred it is necessary to examine developmental char- in the most recent common ancestor of acter state distribution within an explicit phy- Nymphaeales )Fig. 6A). Cellular/multiseriate logenetic context )Diggle 1992, Ra€ 1992). chalazal development was resolved as ancestral Phylogenetic interpretation of endosperm and this state was also retained in Amborella, character state transitions among basal angio- the Illiciales clade and the ancestor of all other sperms. The strategy of Floyd and Friedman angiosperms. A transformation to unicellular )2000) was applied to dissecting the three chalazal development occurred in the ancestor endosperm developmental patterns of basal of Nymphaeales and was retained throughout grade ¯owering plants )oblique/cellular, trans- that clade )Fig. 6B). Cellular/uniseriate micro- verse/cellular, and transverse/helobial) into pylar development was resolved as ancestral characters and character states )Table 2). Ini- and this state was retained in all lineages tiation of endosperm development may entail except for Cabombaceae in which there was a division of the primary endosperm nucleus transformation to free nuclear )Fig. 6C). without wall formation )free nuclear), or Ontogenetic evolution and the origin of nuclear division may be followed by formation helobial endosperm in Nymphaeales. Together, of either an oblique cell wall )oblique/cellular) the patterns of character evolution for division or a wall that is transverse )transverse/cellu- of the primary endosperm nucleus/cell, chala- lar). Early development of the chalazal domain zal development, and micropylar development may be cellular/multiseriate, cellular/uniseri- indicate the following scenario for endosperm ate, free nuclear, or involve no additional ontogenetic evolution among the earliest di- nuclear or cellular proliferation )unicellular). verging angiosperms. The endosperm pattern Finally, early development of the micropylar characterized by an oblique, unequal, cellular domain may be uniseriate with one or more ®rst division )that yields imprecise de®nition of cells )cellular/uniseriate), cellular/multiseriate, micropylar and chalazal domains), cellular or free nuclear. These character states were uniseriate development of the micropylar do- parsimoniously optimized onto a simpli®ed main, and cellular multiseriate development of angiosperm phylogeny in order to trace endo- the chalazal domain )Fig. 5A) represents the sperm character evolution in the basal angio- ancestral angiosperm ontogeny that was re- sperm grade )Fig. 6). tained in Amborella, the Illiciales clade, and For division of the ®rst endosperm cell, the most recent common ancestor of all other oblique/cellular was resolved as ancestral and angiosperms. this state was retained in Amborella, the A transformation from an oblique/cellular ®rst division to transverse/cellular occurred in Table 2. Three characters and possible character the most recent common ancestor of Nym- states associated with early endosperm pattern phaeales and resulted in an ontogeny in which formation the micropylar and chalazal domains are precisely determined by the ®rst division. First division Chalazal Micropylar Additionally, chalazal development was trans- development development formed from cellular/multiseriate to unicellu- Oblique/cellular Cellular/ Cellular/ lar )with invasive growth as a haustorial tube). multiseriate multiseriate Those two evolutionary changes produced a Transverse/cellular Cellular/ Cellular/ modi®ed cellular developmental pattern uniseriate uniseriate )Fig. 5B) that was retained in Nymphaeaceae. Free nuclear Unicellular Free nuclear Finally, micropylar development changed Free nuclear from cellular/uniseriate to free nuclear in the 164 S. K. Floyd and W. E. Friedman: Developmental evolution of endosperm

Fig. 6. Three pattern forming endosperm characters mapped onto simpli®ed angiosperm phylogenies. A Division of the primary endosperm nucleus/cell is coded either as oblique/cellular, transverse/cellular, or free nuclear. Oblique/cellular division is resolved as ancestral for angiosperms and was retained in Amborella, the Illiciales clade, and the ancestor of all other angiosperms diverging above the three basal nodes. Transverse/ cellular division evolved in the common ancestor of Nymphaeales. B Chalazal development is coded either as cellular/multiseriate, unicellular/haustorial, or free nuclear. Cellular/multiseriate development is resolved as ancestral for angiosperms and was retained in Amborella, the Illiciales clade, and the ancestor of all other angiosperms diverging above the three basal nodes. Unicellular/haustorial chalazal development evolved in the common ancestor of Nymphaeales. C Micropylar development is coded either as cellular/uniseriate, cellular/ multiseriate, or free nuclear. Cellular/uniseriate development is resolved as ancestral for angiosperms and was retained in Amborella, the Illiciales clade, Nymphaeaceae, and the ancestor of all other angiosperms diverging above the three basal nodes. Free nuclear micropylar development evolved in the common ancestor of Cabombaceae most recent common ancestor of Cabomba- Wunderlich 1959) in Nymphaeales. Further- ceae, resulting in the helobial developmental more, comparative developmental analysis of pattern )with a haustorial, unicellular chalazal endosperm within a phylogenetic context domain) that typi®es that family )Fig. 5C). If shows a clear and unambiguous relationship free nuclear endosperm does occur in Euryale between helobial endosperm and cellular en- as reported by Khanna )1964), it was likely dosperm in Nymphaeales, with the derivation derived from cellular endosperm since Euryale of a helobial pattern from a cellular pattern was resolved as highly nested within Nymphae- through a series of character state changes. aceae )Les et al. 1999). Implications of alternative topologies for the Phylogenetic comparison of endosperm interpretation of endosperm evolution. Al- developmental characters supports the hypoth- though several di€erent phylogenetic analyses esis that a transverse/cellular pattern was agree that Amborella, Nymphaeales, and the intermediate between an ancestral oblique/ Illiciales clade are basal to all other extant cellular pattern and a derived helobial pattern. angiosperms, there is support for di€erent These results indicate that helobial endosperm branching orders of these three lineages )Qiu is neither restricted to the monocot clade as et al. 2000). Some analyses )Barkman et al. has been asserted )Swamy and Parameswaran 2000, Qiu et al. 2000) indicate that Amborella 1962, Swamy and Krishnamurthy 1973) nor is plus Nymphaeales may form a clade that is this pattern transitional between cellular and sister to all other angiosperms. This hypothesis free nuclear )Schnarf 1929, Maheshwari 1950, requires no change in the interpretation of S. K. Floyd and W. E. Friedman: Developmental evolution of endosperm 165 endosperm character polarity or developmen- Gerhart 1998, von Dassow and Munro 1999, tal evolution described above. Graham and Ra€ and Ra€ 2000, Ra€ and Sly 2000). Olmstead )2000) found some support for a Modularity, the compartmentalization of de- rooting with Nymphaeales alone. With this velopment into semiautonomous processes topology, we lose the ability to resolve the )Wagner 1996), has been described as a funda- ancestral state for division of the primary mental feature of biological organisms that is endosperm cell )either oblique or transverse) crucial to the evolution of development and and for chalazal development )unicellular or complex adaptations )Bonner 1988, Gilbert cellular/multiseriate). However, rooting of an- et al. 1996, Wagner and Altenberg 1996, Kir- giosperms with Nymphaeales does not lead to a schner and Gerhart 1998, Ancel and Fontana rejection of the hypothesis that oblique/cellular 2000, Ra€ and Ra€ 2000, Ra€ and Sly 2000). endosperm development is ancestral for ¯ow- Although many studies have shown that plant ering plants, nor does it change the conclusion developmental processes are dissociable )re- that helobial endosperm in Cabombaceae is viewed in Diggle 1992), the concept of modu- derived from a cellular endosperm pattern. larity has been discussed almost entirely within Modularity in endosperm development. We the contexts of animal developmental evolution have shown that endosperm in basal angio- )see Ra€ and Ra€ 2000, Ra€ and Sly 2000) and sperms exhibits a bipolar ontogeny in which evolutionary computation )Wagner 1996, two domains )chalazal and micropylar) under- Rotaru-Varga 1999, Calabretta et al. 2000). go di€erential patterns of development, Early endosperm ontogeny seems to ®t the following an initial cellular or free nuclear model of a modular process, as there are division. Furthermore, these three features individual dissociable components )e.g. chala- )®rst division, micropylar development, and zal and micropylar development) that have chalazal development) have evolved indepen- been the ``modular unit[s] of evolutionary dently in di€erent angiosperm lineages, leading transformation'' )Wagner 1996, p. 38). With- to di€erent developmental patterns )Floyd and out an understanding of the genetic control of Friedman 2000). For example, in the most endosperm development, endosperm develop- recent common ancestor of Nymphaeales, mental characters do not yet fully meet the cellular multiseriate chalazal development )an- genetic criteria of modules de®ned by Ra€ cestral for angiosperms) was transformed to )1996) and Wagner )1996). However, there is unicellular uninucleate chalazal development evidence that micropylar and chalazal endo- )Fig. 6B), without any change in early micro- sperm domains exhibit di€erential gene pylar development )Fig. 6C). During subse- expression in Arabidopsis )Luo et al. 2000) quent evolution of the Nymphaeales clade, and maize )Hueros et al. 1995, Doan et al. micropylar development was transformed 1996, Opsahl-Ferstad et al. 1997, Olsen 1998). from cellular uniseriate to free nuclear in the Molecular genetic analysis of endosperm de- most recent common ancestor of Cabomba- velopment in these model plant taxa, directed ceae )Fig. 6C), but chalazal development did toward revealing the developmental programs not change )Fig. 6B). Thus at least two of the of early ontogeny, are needed to fully examine early, pattern forming stages of endosperm the modular nature of endosperm ontogeny. development )micropylar development and Endosperm is a simple organism with chalazal development) can be described as relatively few developmental modules com- dissociable processes )sensu Ra€ 1996). pared to mature plant sporophytes and cer- Dissociability of the components of ontog- tainly compared to metazoans. Thus eny has long been recognized )Diggle 1992) and endosperm may serve as an ideal organismic forms the foundation for the concept of devel- system in which to study the role of modularity opmental modularity )Ra€ 1996, Wagner 1996, in evolution by determining the genetic control Wagner and Altenberg 1996, Kirschner and of modular development and the types of 166 S. K. Floyd and W. E. Friedman: Developmental evolution of endosperm genetic changes that have led to the transfor- )University of Colorado), the State Botanical mation of modules. Endosperm can also serve Garden of Georgia, and Leonard Thien )Tulane to bring plant development into the discussion University) for providing Illicium, Nuphar,and of modularity which is important if the concept Amborella specimens. We also thank Pamela Diggle is to remain a general theme in evolutionary )University of Colorado) and John Herr )University biology. Studies of simple living organisms like of South Carolina) for valuable suggestions for the improvement of the manuscript. Finally we thank endosperm, along with the emerging data from Tom Lemieux )University of Colorado), Dr. mathematically-based simulations investigat- Tanguy Ja€re , )Centre IRD, New Caledonia), ing the role of modularity in evolution )e.g. Mr. Papineau )Department of Natural Resources, Gruau 1995; Calabretta et al. 1998a, b; Northern Province, New Caledonia), and the De- Rotaru-Varga 1999; Ancel and Fontana 2000; partment of Natural Resources, Southern Province, Calabretta et al. 2000), have the potential to New Caledonia for assistance with collections of provide signi®cant insights into what is perhaps Amborella in New Caledonia. This work was funded a fundamental process in organismic evolution. by grants from the National Science Foundation )DEB 9701210 to W.E.F. and S.K.F., IBN 9816107 to W.E.F.), the Botanical Society of America, and Conclusions the Department of Environmental, Population, and Organismic Biology, University of Colorado. Analysis of endosperm development in repre- sentatives of the three putative basal angio- References sperm lineages has resulted in a more complete hypothesis for the ancestral ontogeny than Ancel L. W., Fontana W. )2000) Plasticity, evolv- previously proposed, in which oblique/cellular ability, and modularity in RNA. J. Exper. Zool. and transverse/cellular patterns were not rec- 288: 242±283. ognized as distinct character states )Floyd and Barkman T., Chenery G., McNeal J., Lyons-Weiler Friedman 2000). 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Addresses of the authors: Sandra K. Floyd* Boulder, Colorado 80309, USA. *Present )sk¯[email protected]) and William E. Friedman, address: Section of Plant Biology, One Shields Department of Environmental, Population, and Avenue, University of California, Davis, CA Organismic Biology, University of Colorado, 95616, USA.