American Journal of Botany 96(9): 1581–1593. 2009. S EED FERTILIZATION, DEVELOPMENT, AND GERMINATION IN HYDATELLACEAE (NYMPHAEALES): IMPLICATIONS FOR ENDOSPERM EVOLUTION IN EARLY ANGIOSPERMS 1 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. Sokoloff 5 2 Royal Botanic Gardens, Kew, Richmond, Surrey TW9 3AB, UK; 3 The University of Western Australia, Crawley, WA 6009, Australia; 4 Department of Earth Sciences, Royal Holloway University of London, Egham, Surrey, TW20 0EX, UK; and 5 Department 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 fertiliza- tion 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, simi- lar to the chalazal endosperm haustorium of waterlilies. The endosperm condition in Trithuria and waterlilies resembles the helo- bial 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 cha- lazal and micropylar domains, but not their subsequent developmental trajectories. The unusual tissue layer surrounding the bi- lobed 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 identifi ca- Poales. Following detailed systematic study ( Sokoloff et al., tion of the earliest lineages of fl owering plants. Phylogenies 2008a ), Hydatellaceae now consist of a single genus, Trithuria , based on molecular data indicate that although monocots and containing 12 species: 10 from Australia, one from India, and eudicots represent two entirely distinct major angiosperm one from New Zealand. clades, a few taxa do not belong in either lineage and are either Improved resolution of early-angiosperm relationships has early divergent on the angiosperm tree or placed in an unre- enhanced our understanding of “ key ” angiosperm features such solved polytomy with monocots and eudicots ( Fig. 1 ). Though as the early evolution of the fl ower and double fertilization. Un- relatively species-poor, the early-divergent angiosperm lin- derstanding structural homologies and developmental pathways eages assume disproportional signifi cance in comparative stud- in early-divergent angiosperms is important in recognizing how ies of angiosperm evolution. In most analyses, the basal they evolved from nonfl owering ancestors among the gymno- angiosperm grade (termed the ANA grade, formerly the ANITA sperms. Furthermore, embryological characters can be impor- grade), consists of three lineages: Amborellaceae (one genus: tant for understanding systematics among plants in which the Amborella ), Nymphaeales (about eight genera: Barclaya , Bra- sporophyte is highly reduced, including Hydatellaceae. For ex- senia , Cabomba , Euryale , Nymphaea [including Ondinea ], ample, embryological and ovule characters of Hydatellaceae Nuphar , Trithuria , Victoria ) and Austrobaileyales (fi ve genera: (pendulous-anatropous ovule, presence of a starchy perisperm Austrobaileya , Illicium , Kadsura , Schisandra , Trimenia s.l.). and cellular endosperm development) were Hamann ’s (1976) Most recently, Saarela et al. (2007) reassigned the small aquatic primary reasons for segregating them as a family distinct from family Hydatellaceae to the waterlily clade, Nymphaeales, from Centrolepidaceae. Embryological characters in early-divergent its former placement close to the grasses in the monocot order angiosperms have been the subject of numerous recent studies (e.g., Batygina et al., 1980, 1982; Battaglia, 1986 ; Shamrov and 1 Manuscript received 28 January 2009; revision accepted 14 April 2009. Winter, 1991 ; Winter and Shamrov, 1991a , b ; Shamrov, 1998 ; The authors thank R. Bateman for critically reading the manuscript and Batygina and Vasilyeva, 2002 ; Williams and Friedman, 2002 , T. Macfarlane for help with fi eldwork in Australia. F. Marone and M. 2004 ; Friedman and Williams, 2003 ; Friedman et al., 2003 ; Stampanoni (Tomcat Beamline, Swiss Light Source, Paul Scherrer Friedman, 2006 , 2008 ; Tobe et al., 2007 ; Rudall et al., 2008 ; Institute) and S. Joomun (Royal Holloway, University of London) provided Friedman and Ryerson, 2009 ; Williams, 2008 , 2009 ). Friedman assistance with synchrotron x-ray tomographic microscopy, and the Swiss and Williams (2003) hypothesized that a four-celled, four-nu- Light Source and EU provided time and funding to work there. The research cleate (single-module) gametophyte represents the plesiomor- was partly supported by a 2007 CoSyst grant. 6 Author for correspondence (e-mail: [email protected] ) phic condition in angiosperms and gave rise to the more 7 Present address: Museum of Paleontology, University of Michigan, common seven-celled, eight-nucleate (double-module) condi- 1109 Geddes Road, Ann Arbor, Michigan 48109 USA tion that occurs in more than 80% of angiosperms ( Palser, 1975 ). The four-nucleate condition characterizes all families of doi:10.3732/ajb.0900033 Nymphaeales (including Hydatellaceae) and Austrobaileyales, 1581 1582 American Journal of Botany [Vol. 96 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 (fi xed material) collection at K. Methods— For 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 µ 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 fi ve by fi ve grid, sealed with parafi lm, and placed in an incubator at 13° C. 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 fi xation, to erode the seed coat and allow penetration of the fi xative to structures within the seed. Material for LM or SEM examination was fi xed in FAA (40% formaldehyde solution – glacial acetic acid – 70% ethanol, 10 : 5 : 85) at room temperature for 36 h, then transferred to 70% ethanol. Material for TEM examination was fi xed in Karnovsky ’ s fi xative (2% parafomaldehyde –2.5% gluteraldehyde in 0.05 M phosphate buffer, pH 7.2) at 4° C 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 fi tted with a tungsten – carbide knife. Sections were stained in toluidine blue and mounted in DPX resin (a mixture of distyrene, a plasticizer, and xylene). Optical sections were photo- graphed using a Leitz Diaplan photomicroscope fi tted with a Zeiss (G ö ttingen, Fig. 1. Diagram showing relationships of early-divergent angiosperm Germany) Axiocam digital camera, in some cases using differential interference lineages, based on recent molecular analyses (e.g., Qiu et al., 2000 ; Soltis contrast microscopy (DIC). Some images were merged using Adobe (San Jose, et al., 2000 ; Saarela et al., 2007 ). California, USA) Photoshop. Drawings were made by sketching over photo- graphed images in Powerpoint (Microsoft, Redmond, Washington, USA). in contrast to the monosporic, seven-celled, eight-nucleate con- For SEM, material was dissected in 70% ethanol, then dehydrated through absolute ethanol and critical-point dried using a Autosamdri-815B CPD (Tousi- dition that is very common in other angiosperms, and the some- mis Research, Rockville, Maryland, USA). Material was mounted onto speci- what divergent eight-celled, nine-nucleate condition that occurs men stubs using double-sided tape, coated with platinum using an Emitech in Amborella ( Friedman, 2008 ; Friedman and Ryerson, 2009 ). (Hailsham, UK) K550 sputter coater, and examined using a Hitachi (Tokyo, Following detailed comparative studies of fl ower, megag- Japan) cold-fi eld emission SEM S-4700-II at 1 kV. ametophyte, and seedling development in Hydatellaceae and For TEM, seeds fi xed in Karnovsky ’ s fi xative were transferred into 0.05 M Nymphaeaceae ( Rudall et al., 2007 , 2008 , 2009 ; Sokoloff et al., phosphate buffer, pH 7.2, further fi xed in 1% osmium tetroxide in 0.05 M phos- phate buffer, dehydrated in an ethanol series, and embedded in LR White 2008b ), our aim in this paper is to continue our current morpho- acrylic resin (London Resin Co.,