American Journal of Botany 96(9): 15811593. 2009.

SEED FERTILIZATION, DEVELOPMENT, AND GERMINATION IN HYDATELLACEAE (NYMPHAEALES): IMPLICATIONS FOR

ENDOSPERM EVOLUTION IN EARLY ANGIOSPERMS1

Paula J. Rudall,2,6 Tilly Eldridge,2 Julia Tratt,2 Margaret M. Ramsay,2 Renee E. Tuckett,3 Selena Y. Smith,4,7 Margaret E. Collinson,4 Margarita V. Remizowa,5 and Dmitry D. Sokoloff5
2Royal

Botanic Gardens, Kew, Richmond, Surrey TW9 3AB, UK; 3The University of Western Australia, Crawley, WA 6009, Australia; 4Department of Earth Sciences, Royal Holloway University of London, Egham, Surrey, TW20 0EX, UK; and 5Department of Higher Plants, Biological Faculty, Moscow State University 119991, Moscow, Russia

New data on endosperm development in the early-divergent angiosperm Trithuria (Hydatellaceae) indicate that double fertilization results in formation of cellularized micropylar and unicellular chalazal domains with contrasting ontogenetic trajectories, as in waterlilies. The micropylar domain ultimately forms the cellular endosperm in the dispersed seed. The chalazal domain forms a single-celled haustorium with a large nucleus; this haustorium ultimately degenerates to form a space in the dispersed seed, similar to the chalazal endosperm haustorium of waterlilies. The endosperm condition in Trithuria and waterlilies resembles the helobial condition that characterizes some monocots, but contrasts with Amborella and Illicium, in which most of the mature endosperm is formed from the chalazal domain. The precise location of the primary endosperm nucleus governs the relative sizes of the chalazal and micropylar domains, but not their subsequent developmental trajectories. The unusual tissue layer surrounding the bilobed cotyledonary sheath in seedlings of some species of Trithuria is a belt of persistent endosperm, comparable with that of some other early-divergent angiosperms with a well-developed perisperm, such as Saururaceae and Piperaceae. The endosperm of Trithuria is limited in size and storage capacity but relatively persistent. Key words: angiosperm evolution; embryo; endosperm; Hydatellaceae; seed development; synchrotron; Trithuria; waterlilies.

Recent years have seen considerable progress in identication of the earliest lineages of owering plants. Phylogenies based on molecular data indicate that although monocots and eudicots represent two entirely distinct major angiosperm clades, a few taxa do not belong in either lineage and are either early divergent on the angiosperm tree or placed in an unresolved polytomy with monocots and eudicots (Fig. 1). Though relatively species-poor, the early-divergent angiosperm lineages assume disproportional signicance in comparative studies of angiosperm evolution. In most analyses, the basal angiosperm grade (termed the ANA grade, formerly the ANITA grade), consists of three lineages: Amborellaceae (one genus: Amborella), Nymphaeales (about eight genera: Barclaya, Brasenia, Cabomba, Euryale, Nymphaea [including Ondinea], Nuphar, Trithuria, Victoria) and Austrobaileyales (ve genera: Austrobaileya, Illicium, Kadsura, Schisandra, Trimenia s.l.). Most recently, Saarela et al. (2007) reassigned the small aquatic family Hydatellaceae to the waterlily clade, Nymphaeales, from its former placement close to the grasses in the monocot order
1

Manuscript received 28 January 2009; revision accepted 14 April 2009.

The authors thank R. Bateman for critically reading the manuscript and T. Macfarlane for help with eldwork in Australia. F. Marone and M. Stampanoni (Tomcat Beamline, Swiss Light Source, Paul Scherrer Institute) and S. Joomun (Royal Holloway, University of London) provided assistance with synchrotron x-ray tomographic microscopy, and the Swiss Light Source and EU provided time and funding to work there. The research was partly supported by a 2007 CoSyst grant. 6 Author for correspondence (e-mail: p.rudall@kew.org) 7 Present address: Museum of Paleontology, University of Michigan, 1109 Geddes Road, Ann Arbor, Michigan 48109 USA doi:10.3732/ajb.0900033

Poales. Following detailed systematic study (Sokoloff et al., 2008a), Hydatellaceae now consist of a single genus, Trithuria, containing 12 species: 10 from Australia, one from India, and one from New Zealand. Improved resolution of early-angiosperm relationships has enhanced our understanding of key angiosperm features such as the early evolution of the ower and double fertilization. Understanding structural homologies and developmental pathways in early-divergent angiosperms is important in recognizing how they evolved from nonowering ancestors among the gymnosperms. Furthermore, embryological characters can be important for understanding systematics among plants in which the sporophyte is highly reduced, including Hydatellaceae. For example, embryological and ovule characters of Hydatellaceae (pendulous-anatropous ovule, presence of a starchy perisperm and cellular endosperm development) were Hamanns (1976) primary reasons for segregating them as a family distinct from Centrolepidaceae. Embryological characters in early-divergent angiosperms have been the subject of numerous recent studies (e.g., Batygina et al., 1980, 1982; Battaglia, 1986; Shamrov and Winter, 1991; Winter and Shamrov, 1991a, b; Shamrov, 1998; Batygina and Vasilyeva, 2002; Williams and Friedman, 2002, 2004; Friedman and Williams, 2003; Friedman et al., 2003; Friedman, 2006, 2008; Tobe et al., 2007; Rudall et al., 2008; Friedman and Ryerson, 2009; Williams, 2008, 2009). Friedman and Williams (2003) hypothesized that a four-celled, four-nucleate (single-module) gametophyte represents the plesiomorphic condition in angiosperms and gave rise to the more common seven-celled, eight-nucleate (double-module) condition that occurs in more than 80% of angiosperms (Palser, 1975). The four-nucleate condition characterizes all families of Nymphaeales (including Hydatellaceae) and Austrobaileyales,

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were carried out on T. submersa (HK). Seeds of Nymphaea nouchalii Burm. f. (Fig. 7G) were obtained from the Kew living collections, and seeds of N. lotus L. (Fig. 10) were obtained from the spirit (xed material) collection at K. MethodsFor germination studies, seeds of T. submersa were germinated in the Conservation Biotechnology Unit at Kew. The seeds were sterilized with 0.5% SDICN (sodium dichloroisocyanurate) for 20 min, rinsed in distilled water, then soaked in 5 ppm (14.4 M) gibberelic acid for 24 h. They were pricked out into Petri dishes containing 1/2 Murashige and Skoog medium (Murashige and Skoog, 1962) in a ve by ve grid, sealed with paralm, and placed in an incubator at 13C. Seeds were collected prior to soaking, then at the following intervals: 1, 2, 7, 14, 21, 28, and 31 d after the initial sterilization of the seeds. Seeds collected before 21 d were soaked in concentrated HCl for 2 h before xation, to erode the seed coat and allow penetration of the xative to structures within the seed. Material for LM or SEM examination was xed in FAA (40% formaldehyde solutionglacial acetic acid70% ethanol, 10 : 5 : 85) at room temperature for 36 h, then transferred to 70% ethanol. Material for TEM examination was xed in Karnovskys xative (2% parafomaldehyde2.5% gluteraldehyde in 0.05 M phosphate buffer, pH 7.2) at 4C for 24 h. For light microscopy (LM), material was embedded in Histo-Technovit (Heraeus-Kulzer, Wehrheim, Germany) 7100 resin and sectioned using a Leica (Wetzlar, Germany) RM 2155 rotary microtome tted with a tungstencarbide knife. Sections were stained in toluidine blue and mounted in DPX resin (a mixture of distyrene, a plasticizer, and xylene). Optical sections were photographed using a Leitz Diaplan photomicroscope tted with a Zeiss (Gttingen, Germany) Axiocam digital camera, in some cases using differential interference contrast microscopy (DIC). Some images were merged using Adobe (San Jose, California, USA) Photoshop. Drawings were made by sketching over photographed images in Powerpoint (Microsoft, Redmond, Washington, USA). For SEM, material was dissected in 70% ethanol, then dehydrated through absolute ethanol and critical-point dried using a Autosamdri-815B CPD (Tousimis Research, Rockville, Maryland, USA). Material was mounted onto specimen stubs using double-sided tape, coated with platinum using an Emitech (Hailsham, UK) K550 sputter coater, and examined using a Hitachi (Tokyo, Japan) cold-eld emission SEM S-4700-II at 1 kV. For TEM, seeds xed in Karnovskys xative were transferred into 0.05 M phosphate buffer, pH 7.2, further xed in 1% osmium tetroxide in 0.05 M phosphate buffer, dehydrated in an ethanol series, and embedded in LR White acrylic resin (London Resin Co., Basingstoke, UK). The resin was polymerized in a vacuum oven at 60C for 20 h. The seeds were sectioned using a ReichertJung Ultracut ultramicrotome tted with glass knives. Semithin sections (0.5 m) were stained with toluidine blue and mounted onto glass slides using DPX. Ultrathin sections were mounted onto formvar-coated slot grids, stained using an LKB Bromma 2168 Ultrostainer (Leica, Deereld, Illinois, USA) with lead citrate and uranyl acetate, examined using a JEOL (Tokyo, Japan) JEM-1210 TEM, and photographed using a Kodak (Rochester, New York, USA) Mega view III soft-imaging system. For synchrotron radiation x-ray microtomography (SRXTM), fruit samples were mounted onto brass stubs using polyvinyl acetate glue. Samples were imaged at the TOMCAT beamline, Swiss Light Source, Paul Scherrer Institute, Villigen, Switzerland. Data were acquired using the 10 objectives on an x-ray microscope, and an exposure time of 400420 ms at 9.9 keV. A total of 1501 2048 projections was acquired over 180. Projection data were processed, and corrected sinograms were then used for segment reconstruction. Reconstructed images were processed using Avizo 5.0 (Mercury Computer Systems, Chelmsford, Massachusetts, USA).

Fig. 1. Diagram showing relationships of early-divergent angiosperm lineages, based on recent molecular analyses (e.g., Qiu et al., 2000; Soltis et al., 2000; Saarela et al., 2007).

in contrast to the monosporic, seven-celled, eight-nucleate condition that is very common in other angiosperms, and the somewhat divergent eight-celled, nine-nucleate condition that occurs in Amborella (Friedman, 2008; Friedman and Ryerson, 2009). Following detailed comparative studies of ower, megagametophyte, and seedling development in Hydatellaceae and Nymphaeaceae (Rudall et al., 2007, 2008, 2009; Sokoloff et al., 2008b), our aim in this paper is to continue our current morphological characterization of Hydatellaceae by examining embryo, endosperm, and seed development, including seed germination. In particular, we address whether endosperm development is entirely cellular in Hydatellaceae, as suggested by Hamann (1976) and Hamann et al. (1979), and whether an endosperm haustorium is formed, as in some waterlilies (Cook, 1902, 1906, 1909; Seaton, 1908; Swamy and Parameswaran, 1962; Schneider, 1978; Floyd and Friedman, 2001), and explore whether this new information can shed light on the evolutionary origin of endosperm. A further goal is to examine whether the unusual tissue layer surrounding the bilobed cotyledonary sheath in some species of Trithuria is a belt of persistent endosperm, as suggested by Sokoloff et al. (2008b), or represents an expanded tegmen layer (i.e., derived from the inner integument), as interpreted by Tillich et al. (2007). MATERIALS AND METHODS
MaterialSeveral stages of early postfertilization development were observed in Trithuria cowieana D.D.Sokoloff, Remizowa, T.D.Macfarl. & Rudall (xed material collected by Macfarlane et al., Northern Territory, Australia, 2008), and some stages in T. lanterna (xed material collected by Macfarlane et al., Northern Territory, Australia, 2008), and Trithuria submersa Hook.f. HK indicates material grown in the Conservation Biotechnology Unit at the Royal Botanic Gardens, Kew (K), from seeds collected by Tuckett at Mersa Road swamp, Western Australia 2006. Other species examined for comparison were T. lanterna D.A.Cooke (K: 47115; Dunlop 4740A, Northern Territory, Australia, 1978); Trithuria australis (Diels) D.D. Sokoloff, Remizowa, T.D.Macfarl. & Rudall (Macfarlane 3357 and Hearn, Western Australia, approx. 50 km E of Manjimup, 1999; vouchers at NSW and PERTH); T. lamentosa Rodway (K: 28269; de Malahide s.n., Lake Dobson, Australia, 1966). Seed germination experiments

RESULTS Prefertilization Embryology before fertilization was described in detail by Rudall et al. (2008) and Friedman (2008). Mature embryo sacs of T. submersa are illustrated here (Fig. 2) to show the micropylar position of the egg apparatus and the chalazal position of the central cell nucleus. Stages before seed dispersalEarly development of the endosperm and embryo was observed for Trithuria cowieana (Figs. 3, 4), and some stages were observed in T. australis, T. bibracteata, T. lamentosa, T. lanterna (Fig. 5), and T. submersa. In T. cowieana, very early postfertilization stages were seen in

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Fig. 2. Trithuria submersa, unfertilized four-nucleate embryo sacs. c = central cell, ea = egg apparatus (nuclei not visible), eg = egg cell, m = micropyle, st = stigmatic hair, sy = synergid. Scale bars = 20 m in A, 10 m in BD.

reproductive units (RUs) whose stalks were still extending (Fig. 3G); subsequently, RUs stalks extend farther to bear fruits above the tips of the leaves. Pollen tubes were observed penetrating the micropyle and extending into the embryo sac in T. cowieana (Fig. 3A, C, D). Soon after fertilization, the innermost layer of the inner integument becomes rapidly thickened in the micropylar region to form a tegmen (compare Fig. 3C with Fig. 3D). The zygote (Figs. 3AD, 5E), initially forms two prominent nucleoli, then divides to form a two-celled embryo, each cell possessing two prominent nucleoli (Figs. 4, 5A, B). The proembryo sometimes possesses a clear suspensor cell (Fig. 5C, D). The primary endosperm nucleus (Fig. 3A) lies in the center of the embryo sac and possesses two nucleoli. It undergoes mitosis and a transverse cell wall is formed, resulting in two clear domainschalazal and micropylar. The chalazal haustorial domain contains a single large nucleus (Fig. 3BF) that grows in size until it is extremely large (Fig. 4); the haustorial domain eventually degenerates to form a space (Figs. 6, 7). In the micropylar domain, the single initial nucleus (Fig. 3C) subsequently undergoes several mitoses. At least, the rst division of the micropylar domain is cellular (Figs. 3B, DF, 5E); cell walls are subsequently sometimes difcult to distinguish (Fig. 4), but endosperm is

clearly cellularized at later stages (Figs. 5A, C, D), suggesting that divisions are cellular from the onset in the micropylar domain. Dispersed seeds In dispersed seeds of T. submersa (Fig. 6AD), the embryo consists of a single-celled suspensor and a small, globular proembryo that is attened at one pole, but with no differentiation of tissues or development of cotyledons. The embryo is surrounded by cellular endosperm. At this stage, there is normally a space between the endosperm and perisperm in all species (Figs. 6, 7), though the endosperm maintains contact with the perisperm at the edge of the seed. Perisperm is formed before fertilization and occupies more than 80% of the seed capacity, as also noted by previous authors (Hamann, 1976; Friedman, 2008; Rudall et al., 2008). Perisperm is composed of large cells packed with starch grains. Apart from the peripheral layer, most perisperm cells are arranged in groups; within each group, the cell walls start to break down and the nuclei clump together (Fig. 5F, G). Hydatellaceae share this character (perisperm) in common with other Nymphaeales (Khanna, 1964, 1965; Schneider, 1978, 1983; Schneider and Ford, 1978; Rudall et al., 2008), other early-divergent angiosperms (e.g., Piperaceae: Johnson, 1900, 1902; Saururaceae:

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Fig. 4. (AD) Trithuria cowieana, early postfertilization with diagrams. e = endosperm nucleus in micropylar domain, em = embryo, hn = chalazal (haustorial) nucleus. In diagrams, embryo is in red, micropylar endosperm domain is in yellow, chalazal (haustorial) endosperm domain is in green. Scale bars = 10 m.

Fig. 3. Trithuria cowieana, embryo sacs at earliest postfertilization stages. (A) Pollen tube present, with undivided primary endosperm nucleus and zygote. Red square outlines region where an optical section in a slight different focal plane has been overlaid to show pollen tube. (B) Micropylar endosperm cell has divided again to form two cells; chalazal (haustorial) nucleus remains undivided. (C) Penetrated pollen tube still present in micropyle and tegmen not formed. The embryo sac could be fertilized, in which case the endosperm has undergone one cellular division into chalazal and micropylar domains. However, the tegmen is not yet formed, as in Fig. 1A, so another possible interpretation of this image is that this is an unfertilized double embryo sac of the type that is commonly found in Trithuria (described by Rudall et al., 2008), in which case the lowermost nucleus is another partly formed unfertilized embryo sac, rather than a chalazal endosperm haustorium. (D) Pollen tube still present in embryo sac, micropyle compressed and tegmen forming; micropylar endosperm cell has divided again to form two cells; chalazal (haustorial) nucleus remains undivided. (E, F) Stage slightly later than in D; zygote (proembryo) with two prominent nucleoli. (G) Longitudinal section of top of a single plant showing three reproductive units (RUs) at different developmental stages (youngest at bottom left); the uppermost ovule in the largest RU (arrow) is recently fertilized. e = endosperm nucleus in micropylar domain, em = embryo, hn = chalazal (haustorial) nucleus, p = pollen tube, pe = primary endosperm nucleus, ttl = thickened tegmen layer, z = zygote. Scale bars = 10 m in AD, F, 20 m in E, 100 m in G.

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Fig. 5. (AE) Trithuria lanterna, (E) zygote and (AD) early proembryo development. (F, G) T. cowieana, perisperm, showing clumped groups of nuclei. e = endosperm nucleus in micropylar domain, em = embryo, h = haustorium nucleus, z = zygote. Scale bars = 10 m.

Seaton, 1908), and some monocots (Rudall, 1997), though the perisperm of Acorus, the putative sister to all other monocots, is nonhomologous (Rudall and Furness, 1997). At the micropylar end of the seed is a conspicuously thickened layer of tegmen (inner integument) cells beneath a conical testal structure (the operculum), as in waterliles (e.g., Collinson,

1980). At the chalazal pole, there is a group of densely packed, thick-walled cells. Seed sowing and germinationSeven days after sowing, the operculum remains intact, and the seed has not enlarged, though the embryo has grown slightly (Fig. 8A). After 14 d, the embryo

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Fig. 6. Trithuria submersa, sown, dispersed seeds at (AE) 7 days after sowing, (F, G) 14 d after sowing, (H) 21 d after sowing. e = endosperm, em = embryo, h = haustorial space, o = operculum, p = perisperm, ttl = thickened tegmen layer. Scale bars = 20 m in A, D, E; 50 m in B, C, FH.

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Fig. 7. Synchrotron radiation x-ray microtomographic (SRXTM) images (A, C, EG, longitudinal sections; B, D, transverse sections). (A, B) Trithuria submersa. (C, D) T. occidentalis. (E, F) T. bibractaea. (G) Nymphaea nouchalii. e = endosperm, em = embryo; f = fruit wall, hs = haustorial space; o = operculum, p = perisperm, s = seed coat, ttl = thickened tegmen layer.

has clearly proliferated, causing it to rupture the seed coat and displace the operculum (Figs. 6F, G, 8B, C); the thickened layer of the tegmen protrudes like a ruff (Fig. 8C). The space resulting from degeneration of the chalazal haustorium between the perisperm and the endosperm is reduced in size (Fig. 6F, G) and is eventually obliterated (Fig. 6H) by embryo enlargement. After 21 d, the operculum becomes detached from the seed, and the embryo emerges from the micropylar end (Figs. 6H, 8C). From 28 d after sowing, there is clear polarization of the root and shoot tissues (Figs. 8D, 9AC). The seedling haustorium interfaces with the perisperm via a distinct transfer layer, but a small amount of persistent endosperm tissue remains (Fig. 9D). DISCUSSION Double fertilization Our observations of pollen tubes in the embryo sac of T. cowieana (Fig. 3A, C, D) are a good indicator that double fertilization occurs in Trithuria. No obvious

free sperm nuclei were observed, and synergids (or degenerated synergids) are at best difcult to recognize in Trithuria because of their small size (Friedman, 2008; Rudall et al., 2008). However, the primary endosperm nucleus (Fig. 3A) is relatively large and bears two nucleoli, consistent with a double set of chromosomes (Williams, 2009), the expected (diploid) condition after fusion of a haploid sperm with a haploid central cell nucleus. Diploid endosperm is the common condition in species with a four-nucleate embryo sac, including Nymphaeales and Austrobaileyales (e.g., Williams and Friedman, 2002), and a four-nucleate embryo sac has been observed in several species of Trithuria (Rudall et al., 2008). Triploid endosperm is the more common condition in angiosperms, but this condition results from fusion between a sperm and a diploid fusion nucleus (or two polar nuclei) of a seven-celled embryo sac. Embryo and seed developmentThere are clear similarities between mature seeds of Trithuria submersa and those of some

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Fig. 8. Trithuria submersa, SEM sown seeds. (A) Dispersed seed after 24-h soak in gibberelic acid, (B) 14 d after sowing, (C) 21 d after sowing, (D) 28 d after sowing. op = operculum. Scale bars = 50 m.

waterlilies, including the presence of a well-developed tegmen at the micropylar end of the seed, a highly characteristic operculum, a large starchy perisperm that occupies most of the seed volume, and a small endosperm that persists around the embryo after germination (e.g., Schneider, 1978; Schneider and Ford, 1978; Collinson, 1980; present study). This combination of featuresfour-nucleate embryo sac, operculate seeds with perisperm and haustorial endospermis probably unique to the waterlily clade (Nymphaeales, including Hydatellaceae) among angiosperms. These features are therefore potentially a useful identication tool for well-preserved fossil material, though admittedly the embryo sac is rarely preserved, even in permineralized fossil material where perisperm is documented (Cevallos-Ferriz and Stockey, 1989). The minimal degree of embryo development in the dispersed seed of Trithuria resembles the condition in some waterlilies (Fig. 10), though many other waterlilies, such as Nuphar luteum, Nymphaea spp. (including Ondinea), Euryale ferox, and Barclaya longifolia possess a well-differentiated embryo in the dispersed seed (e.g., Meyer, 1960; Valtzeva and Savich, 1965; Schneider, 1978; Schneider and Ford, 1978; Shamrov, 1998), indicating some variation within the Nymphaeales in timing of developmental stages. A chalazal endosperm haustorium occurs in Hydatellaceae We have observed a chalazal haustorium at early stages in numerous specimens of T. cowieana (Figs. 3, 4). This condition compares closely with the condition reported in other Nymphaeales, which possess a chalazal haustorium (discussed later). Previous studies on the apomictic New Zealand species T. inconspicua (formerly Hydatella inconspicua) (Hamann et al., 1979; Friedman, 2008) and the Indian species, Trithuria konkanensis (Gaikwad and Yadav, 2003) did not report a haustorium. Sections of T. inconspicua illustrated by Hamann et al. (1979) and Friedman (2008) show a few-celled proembryo surrounded by some cellular endosperm adjacent to perisperm,

rather similar to some of our images of T. lanterna (Fig. 5A, B). However, it seems likely that the chalazal haustorium is present in all species but is sometimes inconspicuous and difcult to detect at early stages; we found a large (probably haustorial) nucleus in a few specimens of T. lanterna (Fig. 5E). A single cell located at the chalazal pole in T. inconspicua (Friedman, 2008) could be an inconspicuous chalazal haustorium. The presence of a space between the endosperm and perisperm in dispersed seeds of several species of Trithuria examined here (both in histological sections and x-ray optical sections of nonxed dispersed viable seeds: Figs. 6, 7) indicates a haustorium. The shape and location of the space conforms to the chalazal haustorium, which presumably degenerates at an early stage. SRXTM images show this space in species of Trithuria and Nymphaea (Fig. 7), and in seeds of other species with an endosperm haustorium, such as Saururus (S. Y. Smith, unpublished data). Thus, the space is potentially useful as a ngerprint for haustorial endosperm, though in dispersed seeds of Nymphaea the haustorium is highly variable in shape and sometimes absent (Fig. 10); in many species it extends into the perisperm (e.g., Valtzeva and Savich, 1965; Shamrov, 1998). Many (possibly all) waterlilies possess a chalazal endosperm haustorium, including Brasenia, Cabomba, Nymphaea (including Castalia), and Nuphar (Cook, 1906, 1909; Khanna, 1965; Valtzeva and Savich, 1965; Batygina et al., 1982; Shamrov, 1998; Floyd and Friedman, 2000, 2001). In waterlilies, the primary endosperm mitosis is transverse and divides the endosperm into micropylar and chalazal domains. The micropylar domain undergoes division and becomes cellularized, and the chalazal domain remains undivided and acts as a haustorium, sometimes subsequently extending into the maternally derived perisperm tissue (e.g., in Brasenia: Khanna, 1965; Nymphaea: Valtzeva and Savich, 1965; Cabomba: Floyd and Friedman, 2000). Thus, in all Nymphaeales, the mature cellularized endosperm is formed entirely from the micropylar domain.

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Fig. 9. Trithuria submersa, longitudinal sections of germinated seeds, 28 d after sowing (AC, LM; D, TEM). (A) End view (with seedling axis in cross section), (B, C) lateral views (with seedling axis in longitudinal section), (D) lateral view with detail inset. cs = cotyledonary sheath, e = endosperm, fp = rst leaf of seedling plumule; p = perisperm, r = primary root, rttl = remains of thickened tegmen layer, sh = seedling haustorium, tl = transfer layer of seedling haustorium. Scale bars = 50 m.

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Fig. 10. Nymphaea lotus, longitudinal section of seed (haustorium absent). e = endosperm, em = embryo, p = perisperm, ttl = thickened tegmen layer. Scale bar = 100 m.

The chalazal haustorial domain in Trithuria cowieana contains a single large nucleus that is probably polytene (Fig. 4). The large size of this nucleus probably results from chromosome endoreduplication, which is perhaps the commonest form of endosperm polyploidy, producing giant (polytene) nuclei lled with chromosomes in a prophase-like condition (DAmato, 1984). Although it does not extend into the perisperm like some waterlily haustoria, it could function similarly to aid diffusion of nutrients from the perisperm to the endosperm and embryo. Another possible role is regulation of early endosperm development. Endosperm typology and diversicationEndosperm is traditionally classied into three types: nuclear, cellular, and helobial (Vijayraghavan and Prabhakar, 1984), but as many authors have shown (e.g., Floyd and Friedman, 2000, 2001), this typology is unsatisfactory because it masks a complex suite of characters. The nuclear endosperm type has evolved several times in angiosperms, including more than once in early-divergent angiosperms (e.g., in some Piperaceae: Lei et al., 2002). Loss of cellularization is probably generated by activation of a program for cell-cycle arrest in dividing nuclei (Raghavan, 2006). In angiosperms with nuclear endosperm, including the archetypal model organism Arabidopsis (e.g., Brown et al., 1999), the endosperm is a noncellularized coenocyte during early developmental stages, becoming cellularized later in development. Onset of cellularization can also differ between the micropylar and chalazal domains. Floyd and Friedman (2001) highlighted the distinction between noncellularized early divisions in the micropylar domain in Cabombaceae (Cabomba and Bra-

senia) vs. cellularized early divisions in other waterlilies. Our observation of the cellularized condition in Trithuria suggests that the condition in Cabombaceae represents the loss of early micropylar cellularization. The distinction between cellular and helobial endosperm is often unclear, making it difcult to source comparative information, especially in Nymphaeales. Swamy and Parameswaran (1962) narrowly redened the helobial type as representing only cases in monocots in which the micropylar cell is the larger of the two daughter cells that result from the primary unequal endosperm division, and the smaller chalazal cell often remains noncellularized (coenocytic) and haustorial. Both cellular and helobial types have been reported in Nymphaea (Cook, 1902, 1906, 1909; Seaton, 1908). However, the helobial records were reinterpreted as cellular by Swamy and Parameswaran (1962), because either the rst division is more or less equal, or the chalazal (rather than micropylar) cell is the larger of the two. Despite this, Valtzeva and Savich (1965) concluded that endosperm development in Nymphaea resembles the helobial type. In both cellular and helobial types, the primary mitosis is coupled with cytokinesis, but the resulting micropylar and chalazal daughter cells produce domains with differing morphology and developmental trajectories. The early endosperm condition in Trithuria and waterlilies closely resembles the helobial condition that characterizes many monocots. This condition contrasts with that of Amborella and Illicium, in which the primary partitioning cell wall is oblique, and all or most of the mature endosperm is formed from the chalazal domain (Floyd and Friedman, 2000). The precise location of the primary endosperm nucleus governs the relative sizes of the chalazal and micropylar domains, but not their subsequent developmental trajectories. Vijayraghavan and Prabhakar (1984) suggested that in monocots with helobial endosperm, the chalazal domain is often smaller because of the location of the primary endosperm nucleus close to the antipodals. The central cell nucleus in prefertilized embryo sacs is chalazal (Fig. 2). The primary endosperm nucleus in T. cowieana lies in an almost central position or slightly closer to the antipodals (Fig. 3A), and consequently the primary chalazal cell is only slightly smaller than the micropylar one (Fig. 3C). Thus, there is a clear correlation between location and size. However, there is no clear correlation between location and subsequent cellularization. In Amborella, the primary endosperm nucleus is located in the chalazal region, close to the antipodals, and there is no haustorium, both domains ultimately becoming cellularized (Floyd and Friedman, 2001). The role of the chalazal haustorium in Nymphaeales may be taken over by the antipodals in some lineages with an eight-nucleate embryo sac, thus releasing the chalazal endosperm domain for other roles. The function of the antipodals is obscure; postulated roles include a haustorial one (to transfer nutrients from the nucellus/perisperm) or to secrete growth substances that regulate endosperm development (Willemse and Van Went, 1984). If the four-nucleate embryo sac in Nymphaeales (lacking antipodals) is the plesiomorphic condition in extant angiosperms, as plausibly proposed by Friedman and Williams (2003), then the same could be true for a chalazal endosperm haustorium, perhaps associated with a perisperm. More comparative data are needed on early endosperm development in Austrobaileyales, all of which apparently lack antipodals (e.g., Tobe et al., 2007). However, Illicium (Austrobaileyales) does not t this scenario; a haustorium is absent, and endosperm cellularization is complex and not restricted to one domain (Floyd and Friedman, 2001). Similarly, a different set of

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constraints must operate in monocots, some of which possess both a chalazal endosperm haustorium and antipodals (Swamy and Parameswaran, 1962), though Acorus (sister to all other monocots in most recent phylogenies) possesses large antipodals (Rudall and Furness, 1997) and lacks a chalazal endosperm haustorium (Floyd and Friedman, 2000). Persistence and role of cellularized endosperm Our results demonstrate that the unusual tissue layer surrounding the bilobed cotyledonary sheath in seedlings of some species of Trithuria (including T. submersa) is a belt of persistent endosperm, as suggested by Sokoloff et al. (2008b), and not an expanded tegmen layer (i.e., derived from the inner integument) as suggested by Tillich et al. (2007). Interestingly, a closely comparable (possibly homologous) layer of endosperm that persists around the seedling also occurs in some other early-divergent angiosperms with a well-developed perisperm, such as Saururaceae and Piperaceae (Takhtajan, 1988). Thus, the endosperm of Trithuria, though limited in size and storage capacity, is relatively persistent. Its primary role could be as a regulatory tissue to transfer nutrients from the perisperm to the embryo, though this role is ultimately taken over by the seedling haustorium. Endosperm origin Endosperm is a key feature of angiosperms, yet its homologies and evolutionary origin remain enigmatic. Assessments of endosperm evolution have suffered from its heavily typological classication and from the difculties in homologizing endosperm with structures in gymnosperms. There are two main hypotheses for the evolutionary origin of endosperm (discussed by Favre-Duchartre, 1984; Friedman, 2001; Baroux et al., 2002): (1) as the cellular-growth phase of the female gametophyte (heterochronically delayed until after fertilization) or (2) as a monstrous proembryo that fails to develop into a plant. The latter (proembryo) hypothesis is currently widely preferred, but both hypotheses require further review. Our observations of a haustorium in Trithuria, together with records of similar structures in other ANA-grade angiosperms, could support the proembryo hypothesis. One important similarity between the angiosperm embryo and endosperm is that, in both cases, the primary mitosis often results in a micropylar and chalazal cell that ultimately pattern the resulting structures. In the embryo, divisions of the chalazal cell produce most of the embryo. The micropylar cell gives rise to the stalk (suspensor) that attaches it to the seed coat, or sometimes to both the suspensor and a portion of the embryo proper (Natesh and Rau, 1984). As discussed earlier, a chalazal endosperm haustorium, perhaps associated with a perisperm, could represent the plesiomorphic condition in angiosperms, though this requires further testing. If so, a likely role of the chalazal haustorium, in addition to facilitating transfer of nutrients from the perisperm to the embryo, is to regulate early endosperm development of the micropylar cellularized region. In turn, the micropylar cellularized region, which is relatively small but persistent, may act as a nutrient transfer layer from the perisperm to the embryo, until this role is taken over by the epidermis of the seedling haustorium itself. LITERATURE CITED
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