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 dierential 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 buer )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 buer )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