American Journal of Botany 99(6): 1083–1095. 2012.

E MBRYOLOGY IN T RITHURIA SUBMERSA (HYDATELLACEAE) AND RELATIONSHIPS BETWEEN EMBRYO, ENDOSPERM, AND PERISPERM IN EARLY-DIVERGING FLOWERING 1

W ILLIAM E. FRIEDMAN 2,3,5 , J ULIEN B. BACHELIER 2,3 , AND J OSÉ I. HORMAZA 3,4

2 Department of Organismic and Evolutionary Biology, Harvard University, 26 Oxford Street, Cambridge, Massachusetts 02138 USA; 3 Arnold Arboretum of Harvard University, 1300 Centre Street, Boston, Massachusetts 02131 USA; and 4 Department of Subtropical Pomology, Instituto de Hortofruticultura Subtropical y Mediterránea “la Mayora,” (IHSM la Mayora–CSIC–UMA) 29750 Algarrobo–Costa, Málaga, Spain

• Premise of the study: Despite their highly reduced morphology, Hydatellaceae bear the unmistakable embryological signature of , including a starch-rich maternal perisperm and a minute biparental endosperm and embryo. The co-occurrence of perisperm and endosperm in Nymphaeales and other lineages of fl owering plants, and their respective functions during the course of seed development and embryo germination, remain enigmatic. • Methods: Development of the embryo, endosperm, and perisperm was examined histologically from fertilization through ger- mination in fl owers and fruits of submersa . • Key results: The embryo of T. submersa initiates two cotyledons prior to seed maturity/dormancy, and their tips remain in contact with the endosperm throughout germination. The endosperm persists as a single layer of cells and serves as the interface between the embryo and the perisperm. The perisperm contains carbohydrates and proteins, and functions as the main storage tissue. The endosperm accumulates proteins and aleurone grains and functions as a transfer cell layer. • Conclusions: In Nymphaeales, the multiple roles of a more typical endosperm have been separated into two different tissues and genetic entities: a maternal perisperm (nutrient acquisition, storage, mobilization) and a minute biparental endosperm (nutrient transfer to the embryo). The presence of perisperms among several other ancient lineages of angiosperms suggests a modest degree of developmental and functional lability for the nutrient storage tissue (perisperm or endosperm) within seeds during the early evolution of fl owering plants. Finally, we examine the evolutionary developmental hypothesis that, contrary to longstanding assumptions, an embryo-nourishing perisperm along with a minute endosperm may represent the plesiomorphic condition for fl owering plants.

Key words: aleurone; cotyledons; embryo; germination; Hydatellaceae; Nymphaeales; origin of angiosperms; seed; Trithuria submersa .

In the 5 years since members of the Hydatellaceae vaulted reproductive biology present many diffi culties in assessing from the relative obscurity of the to the stardom of the homologies and reconstructing evolutionary history. ancient angiosperm lineage Nymphaeales (Saarela et al., 2007), Considerable study has been focused on the reproductive much work has been focused on the structural and reproductive “units” of the Hydatellaceae and whether they represent infl o- biology of this unusual and important clade of plants ( Rudall rescences with small unisexual fl owers that lack a perianth or a et al., 2007, 2008 , 2009a , b ; Tillich et al., 2007; Friedman, form of transitional protofl owers ( Rudall et al., 2007 , 2009a , b ; 2008 ; Remizowa et al., 2008 ; Sokoloff et al., 2008a , b , 2009 , Saarela et al., 2007 ; Endress and Doyle, 2009 ; Sokoloff et al., 2010 , 2011 ; Carlquist and Schneider, 2009 ; Tratt et al., 2009 ; 2009, 2010 ). The development of the seedlings has also been Taylor et al., 2010; Tuckett et al., 2010a, b ; Prychid et al., 2011; examined but with differing conclusions as to the number of Taylor and Williams, 2012 ; Iles et al., 2012 ). We now know cotyledons present (one or two) ( Tillich et al., 2007 ; Sokoloff that members of the Hydatellaceae represent an extraordinary et al., 2008b ; Rudall et al., 2009b ). Yet, for all of the complexities clade of miniscule wind- or water-pollinated water lilies, whose of understanding the morphology of Hydatellaceae, members highly peculiar reduced morphology and somewhat unusual of this clade bear the unmistakable and unique embryological signature of the Nymphaeales. In retrospect, it is astonishing that given their common structural characteristics, no one had 1 Manuscript received 11 February 2012; revision accepted 12 April speculated that there might be an affi nity of the Hydatellaceae 2012. with water lilies until DNA sequence data revealed their true The authors thank T. Eldridge and S. Holloway for help with histology phylogenetic position. and S. Holloway for earlier versions of some fi gures. They also thank S. In Hydatellaceae, the female gametophyte has now been Renner and an anonymous reviewer for helpful comments. This research shown to match the four-nucleate, four-celled Nuphar/Schisandra was funded by a National Science Foundation grant (IOS–0919986) to type found in all other water lilies (Friedman, 2008 ; Rudall et al., W.E.F. and a grant from the Spanish Ministry of Science and Innovation– European Regional Development Fund (AGL2010–15140) to J.I.H. W.E.F. 2008). The development of operculate seeds with a minimal and J.B.B. contributed equally to the manuscript. diploid endosperm and substantial perisperm is also fundamentally 5 Author for correspondence (e-mail: [email protected]) similar to other water lilies ( Saarela et al., 2007 ; Tillich et al., 2007; Rudall et al., 2007, 2009b ; Friedman, 2008; Sokoloff doi:10.3732/ajb.1200066 et al., 2008b; Tuckett et al., 2010b), except that in Trithuria ,

American Journal of Botany 99(6): 1083–1095, 2012; http://www.amjbot.org/ © 2012 Botanical Society of America 1083 1084 AMERICAN JOURNAL OF BOTANY [Vol. 99 maternal provisioning of the seed is initiated prior to fertiliza- seed maturation/dormancy and from the point when seeds tion, an exception to the temporal pattern of post-fertilization emerge from dormancy through germination and the establish- maternal provisioning to seeds found in all other angiosperms ment of the young seedling. In particular, we pay special atten- ( Friedman, 2008 ). tion to the physiological interactions between the embryo and Recently, the basic structure of developing and germinating its two embryo “support” tissues, the diploid genetically bipa- seeds was characterized in Hydatellaceae ( Rudall et al., 2009b ; rental endosperm and the diploid genetically maternal perisperm. Tuckett et al., 2010b). However, information about the devel- Here we demonstrate that the perispermous seeds of Trithuria opment and the functional interactions between the three dif- exhibit a very defi nite set of functional roles for the minute ferent tissues present in the seeds, the maternally developed diploid endosperm that persists through the time of germina- perisperm (nucellus seed storage tissue), the diploid biparental tion and early seedling emergence. We also discuss possible endosperm, and the embryo, remains sparse in Hydatellaceae evolutionary-developmental transitions that might have occurred ( Yamada et al., 2001 ; Rudall et al., 2007 , 2009b ; Saarela et al., iteratively between sexually formed endosperms and genetically 2007; Tillich et al., 2007; Tuckett et al., 2010b). Hamann (1962, maternal perisperms as they relate to the early diversifi cation of 1975 , 1976 ) described the basic attributes of embryos in Trithuria embryo-nourishing patterns among fl owering plants. (formerly Hydatella; see Sokoloff et al., 2008a) submersa and T . fi lamentosa ( Hamann et al., 1979 ), and Gaikwad and Yadav (2003) performed a similar study for T. konkanensis , but at the MATERIALS AND METHODS time, the true phylogenetic affi nities of Hydatellaceae with water lilies were unknown to these authors. Finally, as is often Embryo development — Trithuria submersa seeds were collected in 2001 in the case in reproductive studies of angiosperms, the critical the wild in various locations in Victoria, Australia, by Andrew Drinnan and phase of development that lies between seed maturation (and William Friedman. Seeds were sown and plants were grown fi rst at the Univer- dormancy) and seed germination has been almost entirely over- sity of Melbourne and subsequently over several generations at the University looked in studies of Hydatellaceae. of Colorado at Boulder and the Arnold Arboretum of Harvard University. For this study, seeds were harvested from cultivated plants at different stages of Despite the homoplasious presence of a substantial perisperm development during the summer of 2010. Collected material was studied in and highly reduced endosperm in groups such as the Nympha- two developmental phases: from fertilization to seed maturity and from seed eales, Piperales, and various monocot and eudicot lineages maturity through germination and seedling establishment. For the fi rst stages, ( Fig. 1 ; Doyle, 1994; Doyle et al., 1994; Rudall and Furness, fl owers and developing seeds were fi xed for 24 h in a modifi ed PIPES buffer 1997 ), little has been written about the specifi c developmental adjusted to pH 6.8 (50 mmol/L PIPES, 1 mmol/L MgSO4 , 5 mmol/L EGTA) relationships of a maternally derived perisperm to the genetically with 4% glutaraldehyde or 4% acrolein (Polysciences, New Orleans, Louisiana, USA). For later developmental stages, seeds were fi rst sown in water at 12 ° C biparental endosperm and embryo during seed provisioning and and collected at weekly intervals over the course of 9 wk, then fi xed in 4% germination (see also Lersten, 2008 ). Moreover, key evolutionary acrolein for 24 h. All fi xed materials were rinsed briefl y a few times with a transitions between embryo-nourishing endosperms and peris- modifi ed PIPES buffer, dehydrated through a graded ethanol series, and stored perms have not been formally analyzed. in 70% ethanol. Our goal herein is to carefully evaluate embryo development in Trithuria submersa, from fertilization through the time of Light microscopy — samples were dehydrated through a graded etha- nol series up to 100%, then infi ltrated and embedded with glycol methacrylate (JB-4 Embedding Kit, Electron Microscopy Sciences, Hatfi eld, Pennsylvania, USA). Embedded materials were serially sectioned into 3 to 5 µm thick ribbons and mounted onto slides. Serial sections were stained with a periodic acid– Schiff’s (PAS) reaction to detect all carbohydrates including starch grains (Schiff’s reagent from Fischer Scientifi c, Pittsburgh, Pennsylvania, USA) and 1% aniline blue-black (ABB, also called amido black; Harleco, Clearwater, Florida, USA) in 7% acetic acid to detect proteins. Alternatively, 0.1% aque- ous toluidine blue O (J.T. Baker, Philipsburg, New Jersey, USA) was used as a nonspecifi c dye or, sometimes, as a counterstain after PAS.

Digital imaging —A Zeiss Axio Imager Z2 microscope equipped with a Zeiss High Resolution Axiocam digital camera (Zeiss, Oberkochen, Germany) was used for bright fi eld and differential interference contrast (DIC) micros- copy and digital imaging. Pictures, line drawings, and fi gures were all pro- cessed and edited using Adobe Creative Suite 5 (Adobe Systems, San Jose, California, USA). Image manipulations were restricted to operations that were applied to the entire image, except as noted in specifi c fi gure legends.

Character analysis —Character evolution was analyzed using the program MacClade 4.03 ( Maddison and Maddison, 2000 ). Terminal taxa in the basal grade of angiosperms are monophyletic families as circumscribed by the An- giosperm Phylogeny Group ( APG III, 2009 ) and placement of the main lin- eages was based on Stevens (2001 onwards). Character states were determined from primary embryological reports. Each trait was coded as known if the depiction of this trait was consistent in the text and the fi gures. Where there was variation for a trait within a family, we determined the ancestral state of the family by parsimony after mapping the character onto a recent phylogeny for the family (traits were considered unordered and unpolarized). In some cases, characters were mapped onto alternative molecular phylogenies. When Fig. 1. Distribution of perisperm among ancient and major angiosperm conclusive polarization could not be achieved, we coded the character state as clades (phylogeny from Stevens, 2001 onwards ). unknown. June 2012] FRIEDMAN ET AL.—EMBRYOLOGY IN TRITHURIA AND PERISPERM EVOLUTION 1085

RESULTS does the subapical cell (yellow in Fig. 3) divide longitudinally to yield two cells ( Figs. 2C, 3E ). These two cells remain undi- Embryo and endosperm development through seed matu- vided until the embryo is comprised of ca. 20 cells (Figs. 2D, rity/dormancy — In Trithuria submersa, the zygote initially re- 3F–I). They then undergo another longitudinal set of divisions mains undivided after fertilization of the female gametophyte, to form a packet of four cells ( Figs. 2E, 3J, 4D, 4I ), that divide while the primary endosperm cell undergoes a limited series of transversally to form a packet of eight cells that are situated cell divisions that yields a multicellular endosperm ( Fig. 4B, G ). between the single suspensor cell and the bulk of the embryo The fi rst division of the zygote is transverse (perpendicular to proper ( Fig. 3K ). the micropylar–chalazal axis of the seed) and gives rise to a The embryo remains globular until just prior to seed dormancy. two-celled proembryo (Fig. 3A). The basal cell of the two- At this point, the embryo initiates two very small cotyledon celled proembryo (red in Fig. 3) undergoes no further divisions primordia (Figs. 2F, 3L; see also Figs. 4J, 5B, 5C, 6B, 6C). At and ultimately differentiates into a single-celled suspensor seed maturity/dormancy, the embryo is thus relatively small ( Figs. 2, 3 ). and dicotyledonous; histologically, it has a well-defi ned proto- Through a series of subsequent cell divisions, the terminal derm and central set of cells that have not yet differentiated into cell (gray in Fig. 3A ) will give rise to the embryo proper ( Figs. 2, ground meristem and procambium ( Figs. 2F, 3L ). 3B–L). A transverse division of the terminal cell yields a linear The fi rst divisions of the fertilized central cell of the female three-celled proembryo (Figs. 3B, 4C, 4H). The subapical cell gametophyte were not observed, but when the zygote is still (yellow in Fig. 3 ) does not immediately divide, whereas the undivided, it is already surrounded by a four-celled endosperm, most apical cell of the three-celled proembryo (green in Fig. 3) indicating that the endosperm of T. submersa is ab initio cellular goes on to form the bulk of the embryo proper (Figs. 2, 3C–L). ( Fig. 4B, G ). When the embryo has reached the three-celled This cell divides longitudinally to create a four-celled proem- stage of development, the endosperm still retains the ovoid bryo (Figs. 2A, 3C). Subsequent longitudinal divisions of the shape of the fertilized female gametophyte ( Fig. 4C, H ). The derivatives of these two most-apical cells form a globular struc- initiation and development of a chalazal haustorial endosperm ture with a clear protoderm (Figs. 2B, 3D). Only after this stage tube, similar to what has been described in other Nymphaeales

Fig. 2. Trithuria submersa . Embryo development. Median longitudinal sections stained with periodic acid–Schiff’s (PAS) reaction. (A) Four- celled embryo (see Fig. 3C). (B) Seven-celled (5 in section) embryo (see Fig. 3D). (C) Eight-celled (six in section) embryo (see Fig. 3E). (D) Approxi- mately 20-celled embryo (see Fig. 3I). (E) Almost mature embryo (see Fig. 3J). (F) Mature embryo with protoderm and two cotyledon primordia (asterisks) (see Fig. 3L). Arrows indicate aleurone grains. Bars = 20 µm. 1086 AMERICAN JOURNAL OF BOTANY [Vol. 99

Fig. 3. Trithuria submersa. Embryo development. Color line drawings of median longitudinal sections of developing seeds, from fertilization through seed maturity/dormancy. (A) Two-celled embryo. (B) Three-celled embryo. (C) Four-celled embryo. (D–I) Globular embryos. (J, K) Nearly mature em- bryos. (L) Mature embryo, with protoderm and two cotyledon primordia (asterisks). Red denotes the basal cell of the proembryo that never divides and differentiates into a single-celled suspensor. Gray denotes the apical cell of the two-celled proembryo that divides into the subapical (yellow) and apical (green) cell of the embryo proper. Yellow denotes the subapical cell of the three-celled embryo and its subsequent derivatives. Green denotes the apical cell of the three-celled proembryo and its derivatives that will form the bulk of the embryo proper, with a clear protoderm (dark green) and a set of undifferenti- ated central cells (light green). Bar = 20 µm.

(see Cook, 1902, 1906 , 1909 ; Seaton, 1908; Meyer, 1960; endosperm during early embryogenesis and later in the suspen- Swamy and Parameswaran, 1962 ; Khanna, 1964a , 1965 , 1967 ; sor and protoderm of the embryo (Fig. 2). However, at seed Valtzeva and Savich, 1965 ; Schneider, 1978 ; Batygina et al., maturity/dormancy, both the endosperm and embryo are devoid 1982 ; Schneider and Jeter, 1982 ; Floyd and Friedman, 2000 , of starch and stain darkly only for proteins ( Figs. 2, 4 ). Numer- 2001 ), was not observed. The cells in the chalazal region of the ous inclusions that we interpret as aleurone grains also accumu- endosperm at the interface with the nucellus appear to divide late in the endosperm ( Fig. 2 ). more slowly and differentiate more quickly than the cells adja- In contrast, starch and proteins begin to accumulate in the cent to the embryo (Fig. 2A, B). The endosperm reaches a max- nucellus of the ovule of T. submersa long before the female imum thickness of three to four cell layers, while the embryo gametophyte is mature and continue to accumulate through becomes globular and begins to appear to crush the immedi- fertilization and up to seed maturity (Fig. 4; see also Friedman, ately surrounding endosperm cells (Fig. 2B–E). At seed matu- 2008; Rudall et al., 2008). The accumulation of starch in the rity, the endosperm consists only of one or two cell layers nucellus is not homogeneous. Three “functional” zones of fl anking the minute embryo ( Fig. 2F ). nucellar tissue can be distinguished: a transient uppermost zone, which caps the female gametophyte, a transient intermediate Nutrient-storage in nucellus and perisperm differentiation — zone, which is situated just beneath the female gametophyte, In Trithuria submersa, a few starch grains can be seen in the and a lowermost zone, which encompasses the remainder of June 2012] FRIEDMAN ET AL.—EMBRYOLOGY IN TRITHURIA AND PERISPERM EVOLUTION 1087

Fig. 4. Trithuria submersa. Nutrient accumulation in nucellus and perisperm formation. Median longitudinal sections of female fl owers and seeds, stained with periodic acid–Schiff’s (PAS) reaction (pink) and aniline blue black (blue-green). Red boxes in panels A–E are magnifi ed in panels F–J. Dif- ferentiation of a perisperm into histologically distinct upper and lower zones is apparent from anthesis onward. (A, F) Female fl ower around anthesis, with egg cell and central cell of mature four-celled/four-nucleate female gametophyte. (B–D, F–I) Developing seeds from fertilization to seed maturity. (B, G) Female fl ower recently fertilized, with zygote at center-top of the female gametophyte, fl anked by two (visible) of four endosperm cells and nuclei. (C, H) Seed with three-celled embryo and two-cell-layered endosperm. (D, I) Seed with globular circa 20-celled embryo and three-cell-layered endosperm. (E, J) Seed 2 wk after sowing in water, with embryo and two cotyledon primordia (asterisks) slightly bigger than at maturity/dormancy (see Fig. 2F ). Abbreviations: cc, central cell; egg, egg cell; emb, embryo; end, endosperm; lper, lower perisperm; syn; synergid; uper, upper perisperm; zyg, zygote. Bars = 100 µm (A–E), 50 µm (F–J). the nucellus down to the chalaza ( Fig. 3 ). The uppermost zone derived nucellar tissue, as in other Nymphaeales. This key storage is one to two cell layers thick and largely devoid of starch tissue is traditionally referred to as a perisperm and constitutes throughout the process of seed maturation ( Fig. 4A, F ). After most of the volume of the seed in T. submersa ( Fig. 4 ). fertilization, it is the fi rst zone to be crushed by the growth of the embryo and endosperm ( Fig. 4B, C, G, H ). The transient Embryo development and interactions between endosperm intermediate zone (upper perisperm) is three to four cell lay- and perisperm during the process of germination — During the ers thick and accumulates starch grains prior to fertilization fi rst 2 wk after T. submersa seeds were placed in water to ger- and during early embryogenesis. After fertilization, this zone minate, no conspicuous changes were observed in the embryo, quickly becomes devoid of proteins (which are inferred to be the endosperm, or the perisperm. Only after 2 wk do cells of the mobilized into the endosperm and embryo) and remains fi lled embryo, and especially its cotyledon primordia, start to prolif- with very small starch grains that also progressively disappear erate ( Fig. 4E, J ). As soon as the third week, embryo and coty- (and again are inferred to be mobilized into the endosperm and ledon expansion results in the obliteration of the suspensor and embryo) during embryogeny ( Fig. 4A–D, F–I ). The lowermost the displacement of the operculum of the seed coat ( Fig. 5A ). zone (lower perisperm) is the largest and most distinctive zone The elongation of the two cotyledons serves to push the embryo of the nucellus ( Fig. 4 ). It accumulates starch grains in clusters (root pole fi rst) out of the seed coat. As the embryo emerges, its of cells, which become multinucleate (the nuclei aggregate in bipolar nature becomes increasingly apparent, while the asym- clumps), as reported in an earlier study ( Rudall et al., 2008 , but metric development of the connate bases of the cotyledons tilts see also Rudall et al., 2009b for an alternative interpretation). the embryo away from the longitudinal axis of the seed (Fig. 5A). In contrast with the (transient) intermediate zone, the lower- Once fully emerged from the seed (except for the cotyledons), most zone of nucellus stains darkly for the presence of proteins the embryo starts to elongate at right angles to the longitudinal throughout development up to seed maturity, although it axis of the seed as the fi rst epicotyledonary leaf differenti- lacks the inclusions (aleurone grains) found in the endosperm ates. The expanded bases of the cotyledons form a cotyledon- (Fig. 4). ary sheath ( Fig. 6 ). At seed maturity, most of the carbohydrate and protein reserves As the embryo and its cotyledons develop, the cotyledons in T. submersa are thus stored in the persistent and maternally crush the immediately surrounding endosperm tissue, except 1088 AMERICAN JOURNAL OF BOTANY [Vol. 99

Fig. 5. Trithuria submersa . Germinating seeds 18 d after sowing. (A) Median longitudinal section stained with periodic acid–Schiff’s (PAS) reac- tion and aniline blue black. (B–E) Transverse sections of a single seed stained with PAS and toluidine blue, arranged from top to bottom. The com- parable levels of these transverse sections are noted on the left side of the longitudinal section in (A). By this point in development, the upper perisperm has largely been crushed. (B) Embryo surrounded by united bases of the cotyledons, a single layer of endosperm, and seed coat. (C) Em- bryo root–shoot axis and two cotyledon tips (asterisks) associated with endosperm, and perisperm. (D) Transition zone between endosperm and perisperm. (E) Perisperm. Abbreviations: cots, cotyledons; emb, embryo; end, endosperm; lper, lower perisperm; obt, obturator; sc, seed coat; uper, upper perisperm. Bars = 100 µm.

for a single persistent cell layer at the interface between the DISCUSSION embedded cotyledons and the perisperm ( Figs. 4E, 4J, 5–7 ). Al- though the cotyledons are basally united ( Figs. 5B, 6B ), their Embryo development in Trithuria and other ancient angio- tips are free and remain in tight contact with the endosperm sperm lineages — In Trithuria submersa, the development and throughout the germination process ( Figs. 5A, 5C, 6A, 6C ). expansion of the embryo prior to seed dormancy is limited. This During germination and the emergence of the embryo from the is fundamentally similar to what has been reported in all Hyda- seed, starch grains in the upper perisperm progressively disap- tellaceae ( Hamann, 1975, 1976 ; Hamann et al., 1979 ; Gaikwad pear ( Figs. 5, 6 ). The constituent carbohydrates are assumed to be and Yadav, 2003 ; Rudall et al., 2009b ) and other water lilies transferred by the persistent layer of the endosperm to the em- studied to date (Chiffl ot, 1902 ; Cook, 1902 , 1906 , 1909 ; Con- bryo via the tips of its cotyledons (Figs. 5–7). Thus the endosperm ard, 1905; Seaton, 1908; Guttenberg and Müller-Schröder, acts as a transfer tissue and the embryo never comes into direct 1958; Meyer, 1960; Khanna, 1964a, b ; Valtzeva and Savich, contact with perisperm (maternal sporophyte). Based on the pres- 1965 ; Wheeler Haines and Lye, 1975 ; Schneider, 1978 ; ence of what appear to be aleurone grains in the persistent layer Schneider and Ford, 1978; Schneider and Jeter, 1982; Osborn of endosperm, it is possible that the enzymes responsible for the and Schneider, 1988 ; Shamrov, 1998 ; Floyd and Friedman, mobilization of carbohydrates from the starch reserves of the up- 2000 , 2001 ). In T. submersa (and likely in other species of the per perisperm are derived from the endosperm. Hydatellaceae), a protoderm fi rst differentiates at an extremely During the last phases of seed germination, the elongated early stage of embryo development, but at seed maturity/ embryo develops a fi rst epicotyledonary leaf, and starch re- dormancy, the subdermal cells are not clearly differentiated serves become less dense at the periphery of the perisperm into procambial and ground meristem cells. Cotyledons, re- than in the center ( Figs. 6, 7 ). The single layer of persistent ported here for the fi rst time to be initiated prior to the onset of endosperm is still present between the embryo and the perisperm seed maturity/dormancy in Trithuria , are barely developed. as many as 65 d after sowing (Fig. 7). By this time, the starch This subtle feature of embryo development may well have been and protein reserves are scarce in the perisperm, whose cell missed in previous studies of different Trithuria species where walls are so thin that they are hardly preserved (Fig. 7B). it has been suggested that root and shoot apical meristems are June 2012] FRIEDMAN ET AL.—EMBRYOLOGY IN TRITHURIA AND PERISPERM EVOLUTION 1089

Fig. 6. Trithuria submersa . Germinating seeds 24 d after sowing. (A) Median longitudinal section stained with periodic acid–Schiff’s (PAS) reaction and aniline blue black. (B–E) Transverse sections of a single seed stained with PAS and toluidine blue, arranged from top to bottom. The comparable levels of these transverse sections are noted on the left side of the longitudinal section in (A). (B) Tightly appressed cotyledons within the seed coat. (C) Two coty- ledon tips (asterisks) associated with endosperm and perisperm. (D) Transition zone between endosperm and perisperm. (E) Lower perisperm. Abbre viations: cots, cotyledons; end, endosperm; lper, lower perisperm; sc, seed coat. Bars = 200 µm (A), 100 µm (B–E). not initiated until after germination (e.g., Rudall et al., 2009b some species of Hydatellaceae with the two united cotyledons of and Tuckett et al., 2010b ). , as suggested by Sokoloff et al. (2008b) . Compared with Trithuria , the embryos of Nymphaeaceae A relatively small embryo at seed maturity/dormancy, as found and typically progress further through the histo- in Trithuria and other Nymphaeales, is also common in other early- genetic process of initiating the three primary meristematic tis- diverging lineages of fl owering plants, including and sue systems (protoderm, ground meristem, and procambium) members of the Austrobaileyales ( Martin, 1946 ; Bailey and Swamy, prior to the onset of seed dormancy/maturity. This is also true 1948; Morat and MacKee, 1977; Endress, 1980; Saunders, 1998; of the organogenetic processes of establishing a clear bipolar Floyd and Friedman, 2001 ; Baskin and Baskin, 2007a ; Tuckett et al., embryo axis with root and shoot apical meristems, conspicuous 2010b ; Chien et al., 2011 ). These types of small and relatively cotyledons, and in some cases the fi rst epicotyledonary leaf(ves) undifferentiated embryos require a signifi cant amount of additional (in Cabombaceae: see Cook, 1906; Schneider and Jeter, 1982; growth and development prior to germination and thus correlate Osborn and Schneider, 1988 ; Floyd and Friedman; 2000 ; in with a delay in seedling emergence (Martin, 1946; Baskin and Nymphaceae: see Chiffl ot, 1902 ; Cook, 1902 , 1906 ; Conard, Baskin, 2004 ; Baskin and Baskin, 2007b ; Tuckett et al., 2010b ). 1905 ; Guttenberg and Müller-Schröder, 1958 ; Meyer, 1960 ; Small “underdeveloped” embryos appear to represent the an- Khanna, 1964a , b ; Valtzeva and Savich, 1965 ; Wheeler Haines cestral condition in fl owering plants (Forbis et al., 2002), and and Lye, 1975; Schneider, 1978; Schneider and Ford, 1978; exceptions to this pattern are rare among early-divergent angio- Shamrov, 1998 ; Floyd and Friedman, 2001 ). sperm lineages, having only been reported in three species of There is an extensive literature debating the number of cotyle- Nymphaea (Baskin and Baskin, 2007a). The main difference in dons (one or two) initiated in the embryos of members of the the degree of embryo development at the time of seed dormancy Nymphaeales (see Tillich et al., 2007; Sokoloff et al., 2008b; among water lilies and other early-diverging lineages is thus Rudall et al., 2009b ). Our study clearly shows that two cotyledon likely to be the result of heterochronic shifts in the timing of primordia are initiated in Trithuria submersa and that the cotyle- onset of seed maturity/dormancy relative to the trajectory of dons are congenitally united at their bases as they develop. Further- embryo growth and differentiation (see also Rudall et al., 2009b). more, after rupture of the operculum in T. submersa , development of the two cotyledons is fundamentally similar to what has been Endosperm development and nutrient storage in Trithuria described in other water lilies ( Schneider, 1978 ; Wheeler Haines and other ancient angiosperm lineages— In Trithuria sub- and Lye, 1975; Tillich, 1990). Our study thus supports the ho- mersa, the embryo is surrounded by a small cellular endosperm mology of the cotyledonary sheath surrounding the seedling in in mature seeds, as is the case in all other Hydatellaceae and 1090 AMERICAN JOURNAL OF BOTANY [Vol. 99

Fig. 7. Trithuria submersa . Young seedlings. Median longitudinal sections. (A) Seedling 27 d after sowing, stained with periodic acid–Schiff’s (PAS) reaction. (B) Seedling 65 d after sowing stained with PAS and aniline blue black. Abbreviations: cots, cotyledons; asterisks also denote cotyledon tissue; end, endosperm; lper, lower perisperm. Bars = 200 µm, 50 µm (insets). water lilies studied to date ( Chiffl ot, 1902 ; Cook, 1902 , 1906 , in Trithuria is haustorial (Rudall et al., 2009b). We found no 1909 ; Conard, 1905 ; Seaton, 1908 ; Guttenberg and Müller- evidence in their work or ours of a true endosperm haustorium Schröder, 1958 ; Meyer, 1960 ; Khanna, 1964a , b , 1967 ; Valtzeva that penetrates into the perisperm tissue. Further, Rudall et al. and Savich, 1965; Wheeler Haines and Lye, 1975; Schneider, (2009b) suggest that a “haustorial space” (observed in fi xed 1978; Schneider and Ford, 1978; Schneider and Jeter, 1982; material and synchrotron radiation x-ray microtomographic im- Osborn and Schneider, 1988 ; Shamrov, 1998 ; Floyd and Friedman, ages) between the perisperm and the endosperm that immedi- 2000, 2001 ; Rudall et al., 2009b; Tuckett et al., 2010b). Al- ately surrounds the embryo may be the remnants of a degenerated though we did not directly observe the fi rst divisions of the en- chalazal haustorial endosperm cell. While we did occasionally dosperm in Trithuria , Rudall et al. (2009b) have defi nitively observe a “gap” between the perisperm and the endosperm in documented that it is ab initio cellular as is also the case in most the present study, this was almost always associated with poor Nymphaeaceae ( Cook, 1906 ; Meyer, 1960 ; Schneider, 1978 ; histological preservation of the seed. In most of our material, Schneider and Ford, 1978 ; Schneider and Jeter, 1982 ; Floyd the endosperm appears turgid and healthy and remains in direct and Friedman, 2001 ; Khanna reported a free nuclear endosperm contact with the perisperm (Figs. 4–7). It is unclear whether the in Euryale ferox [Khanna, 1964a, b ] and a helobial endosperm lack of a chalazal endosperm haustorium is an apomorphy of in Nymphaea stellata [ Khanna, 1967 ], but these reports require Hydatellaceae or is plesiomorphic for all extant Nymphaeales, verifi cation [ Floyd and Friedman, 2001 ]). In contrast, in Cabom- with the innovation of this pronounced chalazal endosperm baceae, endosperm development is helobial ( Cook, 1906 ; Floyd haustorium in the common ancestor of Cabombaceae and and Friedman, 2001 ; Khanna [1965] reports a cellular en- Nymphaeaceae ( Floyd and Friedman, 2001 ). dosperm in schreberei , but this report also requires In T. submersa , our study clearly shows that at seed dormancy/ independent verifi cation). However, in both Nymphaeaceae and maturity the endosperm does not contain starch, as is also the Cabombaceae, early endosperm development is typically asso- case in all Nymphaeales and other early-diverging angiosperms ciated with the differentiation of a tube-like chalazal domain studied to date (Floyd and Friedman, 2000 ). In all Nymphaeales, that penetrates into the nucellus and appears to function as a however, starch is accumulated in the nucellus, which is not haustorium during embryogenesis ( Cook, 1902 , 1906 , 1909 ; entirely consumed by the development of the endosperm but Seaton, 1908; Meyer, 1960; Swamy and Parameswaran, 1962; rather differentiates into a perisperm that fi lls up most of the Khanna, 1964a, 1965 , 1967 ; Valtzeva and Savich, 1965; Baty gina mature/dormant seed ( Khanna, 1964a , b , 1965 ; Meyer, 1960 ; et al., 1982 ; Schneider, 1978 ; Schneider and Jeter, 1982 ; Floyd Schneider, 1978 ; Schneider and Ford, 1978 ; Osborn and and Friedman, 2000 , 2001 ). In Hydatellaceae, while a tubular Schneider, 1988; Floyd and Friedman, 2000, 2001 ). Similar de- chalazal extension of the endosperm does not form, it has been velopment of a copious starchy perisperm and a minute en- argued that the single most chalazal cell of the early endosperm dosperm has also been reported in a few other relatively ancient June 2012] FRIEDMAN ET AL.—EMBRYOLOGY IN TRITHURIA AND PERISPERM EVOLUTION 1091 angiosperm families, such as Piperaceae and Saururaceae in endosperm in the seeds of such taxa might represent a “primitive Piperales ( Johnson, 1900a , b , 1902 ; Lei et al., 2002 ). Strikingly, transitional state” in angiosperms was fi rst briefl y noted (with Johnson (1900a , b , 1902 , 1914 ) reported that in Saururus , Pep- attribution to a personal communication from David Haig) by eromia, and Piper , the endosperm is devoid of reserves and Doyle ( Doyle, 1994 ; Doyle et al., 1994 ). Accordingly, the contains aleurone or protein grains, which could facilitate perisperm-based embryo-nourishing behavior, as found in the the mobilization of starch from the perisperm to the embryo. Nymphaeales, would have preceded the evolution of a full- He also noted that a single layer of endosperm is persistent fl edged endosperm as the primary nutrient storage tissue within throughout seed development and seedling germination ( Johnson, a fl owering plant seed. As such, the minute (diploid) endosperm 1902). As in Trithuria, at no point is the embryo found in direct of Hydatellaceae and other water lilies would represent a “pro- contact with the genetically maternal perisperm. toendosperm” that has yet to acquire the functions of nutrient acquisition, storage and mobilization. In the ensuing analysis, Distribution and evolution of perisperm in Nymphaeales we will examine the functional and evolutionary implications and other ancient lineages of angiosperms — Nutrients within of the presence of a minute endosperm and copious perisperm a mature/dormant fl owering plant seed may be stored in one or in Nymphaeales within the context of the alternative hypotheses more of three entities: an endosperm (formed from the second that perisperm in water lilies is apomorphic or is a manifesta- fertilization event), an embryo, and/or a perisperm, a geneti- tion of the plesiomorphic condition for all angiosperms ( Fig. 8 ). cally maternal tissue derived from the nucellus. These angio- sperm patterns of seed provisioning contrast with all other Transitions in endosperm and perisperm function in groups of seed plants, in which the haploid female gametophyte Nymphaeales — The endosperms of most fl owering plants (at (and often the embryo too) is the primary nutrient-storing entity least where the perisperm or the embryo is not the primary nutri- within the dormant seed. ent storage entity within the mature seed) can be viewed as carry- It has long been assumed that the common ancestor of extant ing out a set of four related, but separable physiological missions: angiosperms formed a full-fl edged nutrient-storing and embryo- the acquisition of nutrients from the maternal sporophyte, the nourishing endosperm (Sargant, 1900). Recent evolutionary storage of these nutrients prior to seed dormancy, the mobiliza- analyses ( Forbis et al., 2002 ; Baskin and Baskin, 2007a; Tuckett tion of nutrients after the initiation of germination, and the trans- et al., 2010b ) indicate that embryos of ancient angiosperm fer of these nutrients to the embryo. This pattern characterizes the lineages are small and “underdeveloped” (although we would endosperms of seeds of Amborella (Floyd and Friedman, 2000, argue that an evolutionary perspective suggests that embryos in 2001 ), certain members of the Austrobaileyales (Floyd and derived angiosperms are “overdeveloped”) and hence are not Friedman, 2000 , 2001 ; Yamada et al., 2003 , 2008 ; Chien et al., (cannot be) involved in signifi cant nutrient storage within the 2011 ), and likely most other ancient angiosperm lineages. mature seed. Thus, the presence of either perisperm or the em- If perisperm is an apomorphy of the Nymphaeales (and a full- bryo as the primary storage tissue within the seeds of many fl edged embryo-nourishing endosperm is plesiomorphic for an- distantly related lineages of fl owering plants is thought to be giosperms), it is tempting to describe the loss of three endosperm derived and to have evolved many times over the course of an- biological processes (nutrient acquisition, storage, and mobiliza- giosperm history (e.g., Martin, 1946 ; Cronquist, 1988 ; Donoghue tion) and their assumption by the nucellus (perisperm) as a “trans- and Scheiner, 1992 ; Forbis et al., 2002 ). Remarkably, although fer” of function during the early evolution of the Nymphaeales. the formation of perisperms coupled with highly reduced en- This, however, is problematic, since a “transfer” of function dosperms in diverse angiosperm lineages has been known of for ought to occur within a single genetic (organismal) entity. In the over a century (Johnson, 1902; Humphrey, 1896), there has case of the origin of a derived perisperm and its newly acquired been little analysis of the evolutionary origin of a perisperm and functions (nutrient acquisition, storage, and mobilization) in the its effect on the functional relationships and roles of a tradi- common ancestor of Nymphaeales, the corresponding loss of tional endosperm in fl owering plants. these functions occurs within an endosperm that is genetically Our survey of the embryological literature fi nds that mem- (and organismically) distinct from the maternal sporophyte. Thus, bers of many of the most ancient lineages of angiosperms in the perisperm of Trithuria and other water lilies, the maternal (Nymphaeaceae, Cabombaceae, Hydatellaceae, possibly Tri- sporophyte has taken over the former physiological interactions meniaceae and Ceratophyllaceae, Saururaceae, Piperaceae, between the maternal sporophyte and the endosperm when it Hydnoraceae, Acoraceae) form an embryo-nourishing perisperm comes to nutrient storage and allocation. along with a minute (dozens of cells) or modest-sized en- In light of the putative loss of so many of the basic functions dosperm (e.g., Hallier, 1901 ; Johnson, 1902 ; Cocucci, 1976 ; of endosperm in the common ancestor of Nymphaeales (again, Rudall and Furness, 1997 ; Prakash and Bak, 1982 ; Floyd and assuming that perisperm is an apomorphy of Nymphaeales), it Friedman, 2000; Lei et al., 2002; Shamrov, 2006; Lersten, is striking that endosperm still has a critical role to play in the 2008). In Nymphaeales, the perisperm is paired with a diploid perispermous seeds of Trithuria (and other water lilies). What endosperm, while in other groups, the perisperm is paired with seems noteworthy is the fact that even though the functions of a triploid or even higher ploidy endosperm. nutrient acquisition, nutrient storage, and nutrient mobilization Interestingly, when the presence of a perisperm is mapped onto are not carried out by the endosperm in Trithuria, but instead a current phylogeny of the major lineages of fl owering plants are part of the functions of the nucellus, the endosperm has re- (e.g., Stevens, 2001 onwards ), a noteworthy result is obtained: tained a critical role in seed development and germination, as perisperm either evolved several times during the early diversifi - the transfer tissue (perhaps physiological gatekeeper) for nutri- cation of angiosperms or was the plesiomorphic condition for ents into the embryo (see Humphrey, 1896 ). This is borne out angiosperms as a whole. It is only one step less parsimonious to by the fact that long after germination has begun in Trithuria , a assume that the perisperm and minute endosperm of Nymphae- single layer of endosperm cells always surrounds the embryo so ales represents the ancestral character state for angiosperms. The that the embryo is never in contact with the maternal diploid possibility that the presence of perisperm along with a small perisperm tissue. 1092 AMERICAN JOURNAL OF BOTANY [Vol. 99

Fig. 8. Hypothetical transitions from a haploid embryo-nourishing female gametophyte in gymnosperms to the typical triploid embryo-nourishing endosperms of most fl owering plants. With the recent discoveries of diploid endosperms in Nymphaeales and Austrobaileyales, the triploid endosperms of most angiosperms are likely to be derived from a diploid condition in early fl owering plants. (A) Traditionally, it has been assumed that angiosperm clades with a minute endosperm and a copious perisperm are derived from ancestors with full-fl edged endosperms. If this is the case, the origin of a large perisperm in the common ancestor of Nymphaeales would have entailed a commensurate reduction of a copious diploid endosperm (present in the fi rst angiosperms) to a minute diploid endosperm. The evolution of the typical triploid endosperm in the common ancestor of most other angiosperms would have resulted separately from the addition of a haploid maternal nucleus (second polar nucleus) to the second fertilization event. (B) Alternatively, the fi rst angiosperms may have acquired a minute diploid “protoendosperm,” along with a maternal perisperm that served as the primary embryo-nourishing tissue within the seed. If this is the case, the condition in Hydatellaceae and other water lilies (minute diploid endosperm and a perisperm) would represent the ancestral condition for fl owering plants. The subsequent gain of size and embryo-nourishing function by a diploid endosperm, accompanied by the loss of perisperm would be derived (this condition is found in Illiciaceae and Schisandraceae of the Austrobaileyales), and the origin of a large triploid endosperm would then have occurred in the common ancestor of most other angiosperms. In either evolutionary scenario (A or B), the perisperms of many other lin- eages of fl owering plants are clearly derived through loss of size and function of a copious endosperm.

The persistence of a single or extremely thin layer of en- many, if not all, perispermous taxa, is suggestive of a devel- dosperm, as in Trithuria, has been noted in other clades of opmental and physiological constraint (see Humphrey, 1896 angiosperms with perisperm, including diverse members of for a brilliant early insight into this phenomenon). Thus, in the Piperales (Johnson, 1900a, b , 1902 , 1914 ), Acorus (Buell, angiosperm clades where a perisperm has been acquired (in- 1935), some Zingiberales (Humphrey, 1896), and some cluding potentially, Nymphaeales), transfer of function has Caryophyllales ( Johnson, 1902 ). These studies suggest that resulted in a seed biological system where two tissues the extreme reduction of endosperm in perispermous seeds (maternal perisperm and biparental endosperm) now perform does not imply the loss of all critical endosperm functions. the ancestral combined roles of a single tissue (endosperm) More speculatively, the retention of a viable endosperm in (Fig. 8A). June 2012] FRIEDMAN ET AL.—EMBRYOLOGY IN TRITHURIA AND PERISPERM EVOLUTION 1093

Transitions in endosperm and perisperm function in the embryo-nourishing strategies of members of the Austrobai- Nymphaeales, assuming that perisperm is plesiomorphic leyales, Ceratophyllum , and other ancient fl owering plant for all angiosperms— For over a century, the key intermedi- lineages. ate steps involved in the transition from an embryo-nourish- ing female gametophyte (as in gymnosperms) to the LITERATURE CITED embryo-nourishing triploid endosperm of most fl owering plants have remained obscure. With the recent discovery of APG III [ANGIOSPERM PHYLOGENY GROUP III]. 2009 . An update of the diploid endosperms in ancient lineages of angiosperms (Wil- Angiosperm Phylogeny Group classifi cation for the orders and fami- liams and Friedman, 2002 , 2004 ; Friedman and Williams, lies of fl owering plants: APG III. Botanical Journal of the Linnean 2003; Friedman et al., 2003, 2008 ; Friedman, 2008), one of Society 161 : 105 – 121 . B ACHELIER , J. B. , AND W. E. FRIEDMAN . 2011 . 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