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American Journal of Botany 100(5): 906–915, 2013 ; http://www.amjbot.org/ © 2013 Botanical Society of America

American Journal of Botany 100(5): 906–915. 2013.

After the discovery ( Mathews and Donoghue, 1999 ; Parkinson et al., 1999 ; Qiu et al., 1999 ; Soltis et al., 1999 ) that Amborellaceae, Nymphaeales (Cabombaceae, Nymphaeaceae), and Austrobailey-ales (Austrobaileyaceae, Trimeniaceae, Illiciaceae, and Schisan-draceae) were three of the four most ancient lineages of fl owering plants (the other ancient clade being all other angio-sperms, Fig. 1 ), the focus on reconstructing early angiosperm evolutionary history shifted abruptly to these understudied lin-eages. The additional phylogenetic insight that the aquatic family Hydatellaceae was not situated in the Poales, but was a member of the Nymphaeales ( Saarela et al., 2007 ), only heightened in-terest in understanding patterns of biological diversifi cation among the earliest diverging clades of angiosperms (see Friedman et al., 2012 , and references therein). It is not an overstatement to claim that the last fi fteen years of work on Amborella and members of the Nymphaeales and Austrobaileyales has led to the near–global collapse of a century–old set of paradigms concerning

the reproductive features of the earliest angiosperms ( Floyd and Friedman, 2000 , 2001 ; Williams and Friedman, 2002 , 2004 ; Friedman and Williams, 2003 , 2004 ; Friedman et al., 2003 , 2008 ; Rudall, 2006 ; Friedman, 2006 , 2008 ; Tobe et al., 2007 ; Rudall et al., 2008 , 2009 ; Williams, 2008 ; Friedman and Ryerson, 2009 ; see also Friedman et al., 2012 ).

Yet, for all of the recent progress in characterizing the basic biological features of early divergent lineages of fl owering plants, botanists still continue to puzzle over the key evolu-tionary transition from seed plants that nourish their embryos with haploid female gametophyte tissue (gymnosperms) to seed plants that typically nourish their progeny with a sexually formed endosperm tissue (angiosperms). We now know that in contrast with Amborella and most other fl owering plants, which have triploid endosperms, the common ancestor of fl owering plants is likely to have had a four-celled/four-nucleate female gametophyte that yielded a diploid genetically biparental en-dosperm, as is the case in all extant Nymphaeales and Aus-trobaileyales ( Friedman and Ryerson, 2009 ).

In Nymphaeales, the diploid endosperm is minute and almost entirely devoid of reserves, and the main nutrient-storing and embryo-nourishing tissue in the seed is a copious starchy perisperm that is derived from the nucellus (see Friedman et al., 2012 and references therein). In contrast, in most Austrobailey-ales, the diploid endosperm is typically described as being substantial, serving as the primary nutrient-storing and embryo-nourishing tissue ( Austrobaileya , Endress, 1980 ; Yamada et al., 2003 ; Illicium , Hayashi, 1963a ; Floyd and Friedman, 2000 , 2001 ; Kadsura , Hayashi, 1963b ; Schisandra , Hayashi, 1963b ; Kapil and Jalan, 1964 ). Interestingly, previous studies of seeds of

1 Manuscript received 11 December 2012; revision accepted 28 February 2013.

The authors thank J. Bruhl for his help with the collection of plant material, Peter K. Endress for providing plant material, and A. B. Demarest, T. Eldridge, S. Holloway, T. S. McGillivray, R. A. Povilus, and E. I. Scherbatskoy for help with some of the histology. We also thank two anonymous reviewers for their comments. This research was funded by a NSF grant (IOS–0919986) to W.E.F.

4 The authors contributed equally to the manuscript. 5 Author for correspondence (e-mail: ned@oeb.harvard.edu)

doi:10.3732/ajb.1200632

SEED DEVELOPMENT IN TRIMENIA (TRIMENIACEAE) AND ITS BEARING ON THE EVOLUTION OF EMBRYO-NOURISHING

STRATEGIES IN EARLY FLOWERING PLANT LINEAGES 1

WILLIAM E. FRIEDMAN 2,3,4,5 AND JULIEN B. BACHELIER 2,3,4

2 Arnold Arboretum of Harvard University, 1300 Centre Street, Boston, Massachusetts 02131 USA; and 3 Department of Organismic and Evolutionary Biology, Harvard University, 26 Oxford Street, Cambridge, Massachusetts 02138 USA

• Premise of the study: Seeds of most families in the ancient angiosperm lineage Austrobaileyales produce a full-fl edged geneti-cally biparental embryo-nourishing endosperm. However, seeds of fossil and extant Trimeniaceae have been described as having a perisperm, a maternal nutrient-storing and embryo-nourishing tissue derived from the nucellus of the ovule. Because perisperm is also found in Nymphaeales, another ancient angiosperm clade, the presence of a perisperm in Trimeniaceae, if confi rmed, would be congruent with the hypothesis that the fi rst angiosperms used a perisperm in addition to a minute (nutrient-transferring) endosperm.

• Methods: Seed development was studied from fertilization through maturity/dormancy in Trimenia moorei and in maturing fruits of T . neocaledonica .

• Key results: A persistent layer of nucellar tissue surrounds the endosperm but does not contain stored nutrients and does not function as a perisperm. The nutrient-storing and embryo-nourishing tissue in Trimenia seeds is an endosperm, as is the case in all other members of the Austrobaileyales studied to date.

• Conclusion: The absence of a perisperm and the presence of a typical nutrient-storing and embryo-nourishing endosperm in Trimeniaceae may represent the ancestral condition for angiosperms. However, the combination of a copious nutrient-storing and embryo-nourishing perisperm with a minute endosperm, as in Nymphaeales, remains a plausible plesiomorphic condition for angiosperms as a whole. In either case, the developmental and functional biology of the diploid endosperm of Trimenia (and other Austrobaileyales) differs markedly from the diploid endosperm of Nymphaeales, and is fundamentally similar to the triploid endosperms of most other angiosperms.

Key words: Austrobaileyales; basal angiosperms; diploid endosperm; female gametophyte; Nymphaeales; perisperm.

907May 2013] FRIEDMAN AND BACHELIER―EMBRYO-NOURISHING STRATEGIES IN SEEDS OF EARLY ANGIOSPERMS

extinct and extant Trimenia suggest that, as in Nymphaeales, members of this clade produce a perisperm along with a small endosperm ( Prakash, 1998 ; Yamada et al., 2003 , 2008 ). The combinations of different embryo-nourishing tissues (endosperm, perisperm) and levels of endosperm ploidy (diploid or triploid) among members of the Amborellales, Nymphaeales, and Aus-trobaileyales strongly suggest that the embryo-nourishing strat-egies of early fl owering plants were likely much more diverse and evolutionarily labile than has traditionally been assumed.

With this in mind, we have been exploring the verity of the long-held view that the common ancestor of angiosperms ex-clusively depended upon a nutrient-storing endosperm to nour-ish the developing embryo within the seed and that the perisperm of Nymphaeales is apomorphic. As we have recently shown ( Friedman et al., 2012 ), the hypothesis that a perisperm was primarily responsible for nutrient storage and nourishing the embryo in the fi rst fl owering plants is a plausible (although not most parsimonious) evolutionary scenario. As such, the perisper-mous seeds of Nymphaeales might represent this plesiomorphic condition, and endosperm would thus have gradually acquired its dominant nutrient-storing and embryo-nourishing behaviors after the earliest phases of angiosperm diversifi cation ( Friedman et al., 2012 ).

Reconstruction of the developmental characteristics of ovules/seeds of the members of the Austrobaileyales, as well as the common ancestor of Austrobaileyales, is central to the further

Fig. 2. Young ovule of Trimenia moorei during megasporogenesis. (A) Median longitudinal section stained with periodic acid-Schiff’s reagent (PAS). Large arrowheads show zone of starch accumulation in the center of the nucellus. (B) Higher magnifi cation view of tissue within the upper red box in (A). Starch grains are prominent and numerous (small arrows indicate a few starch grains). (C) Higher magnifi cation view of tissue within the lower red box in (A) showing the chalazal end of the nucellus where megasporogenesis occurs. Abbreviations : ch, chalaza; fun, funicle; ii, inner integument; meg, me-gaspore; nuc, nucellus; oi, outer integument; zm, zone of megasporogenesis. Bars = 200 μ m (A) and 20 μ m (B) and (C).

Fig. 1. Phylogenetic relationships of the major clades of ancient lin-eages of fl owering plants (modifi ed from Stevens, 2001 onward ).

908 AMERICAN JOURNAL OF BOTANY [Vol. 100

acetic acid, and 50% ethyl alcohol). Fruits collected in New Caledonia ( T. neo-caledonica ) were fi xed in FAA. Material fi xed in glutaraldehyde was rinsed a few times with the same modifi ed PIPES buffer, dehydrated through a graded ethanol series and stored in 70% ethanol. Material fi xed in FAA was rinsed a few times with 50% ethanol and stored in 70% ethanol. All spirit collections are deposited at the Weld Hill research facility at the Arnold Arboretum of Harvard University.

Light microscopy — Floral buds and fl owers were dissected to collect car-pels. Carpels and seeds from later stages of fruit development were dehydrated through another ethanol series up to 100%, then infi ltrated and embedded with glycol methacrylate (JB−4 embedding kit [Polysciences, Warrington, Pennsylvania, USA]). Embedded materials were mounted and sectioned serially into 4 μ m–thick ribbons using a Microm HM360 rotary microtome (Thermo Fisher Scien-tifi c, Waltham, Massachusetts, USA) with glass knives. Serial sections were mounted onto slides and stained using a periodic acid–Schiff’s reagent (PAS) to detect all insoluble carbohydrates including starch grains (Schiff’s reagent from Fischer Scientifi c, Pittsburgh, Pennsylvania, USA), 1% aniline blue-black (ABB, also called amido black; Harleco, Clearwater, Florida, USA) in 7% ace-tic acid (BDH, London, UK) to detect proteins, and 0.01% Auramine–O (Allied Chemical, New York, New York, USA) in 0.05M Tris (Mallinckrodt Chemi-cals, Phillipsburg, New Jersey, USA) / HCl buffer adjusted to pH 7.2 to detect lipids. Alternatively, 0.1% aqueous toluidine blue O (J. T. Baker, Philipsburg, New Jersey, USA) was used as a nonspecifi c dye, or as a counterstain after PAS.

Digital imaging — A Zeiss Axio Imager Z2 microscope equipped with a Zeiss High Resolution AxioCam digital camera (Carl Zeiss, Oberkochen, Ger-many) was used for bright fi eld, differential interference contrast (DIC), and

evolutionary assessment of the plesiomorphic condition for embryo–nourishing behavior in the earliest angiosperms. Re-grettably, the evidence previously presented for the presence of a perisperm in Trimeniaceae does not appear to be defi nitive. Yet, proper assessment of this key seed biological feature is criti-cal to inferring the evolutionary history of embryo-nourishing strategies during the early diversifi cation of angiosperms. In this paper, we re-evaluate the embryology and seed development of Trimenia moorei (Oliv.) Philipson and T. neocaledonica Baker f. to confi rm or refute the previously reported presence of a perisperm in Trimeniaceae.

MATERIALS AND METHODS

Material collection and fi xation — Floral buds, fl owers, and fruits of Trime-nia moorei were collected in September 2009 in the fi eld from various locations in New South Wales, Australia, by W. E. Friedman, I. R. H. Telford, T. Eldridge, and J. J. Bruhl (see Appendix S1 in Supplemental Data with the online version of this article). Additional fruits were collected by J. J. Bruhl from October through December 2009 and January 2010 (see Appendix S1 in Supplemental Data with the online version of this article). Fruits of T . neocaledonica were collected in the fi eld by P. K. Endress in 1981 in New Caledonia (see Appendix S1). All plant material collected in Australia ( T. moorei ) was fi xed for 24 hours in 4% glutaraldehyde in a modifi ed PIPES buffer adjusted to pH 6.8 (50mM PIPES and 1mM MgSO 4 (BDH, London, UK); 5mM EGTA (Research Organ-ics, Cleveland, Ohio, USA)) or FAA (10% formaldehyde (37%), 5% glacial

Fig. 3. Mature ovule of Trimenia moorei at the time of fertilization. (A) Median longitudinal section stained with PAS and toluidine blue. Large ar-rowheads identify mature tube-like four-celled/four-nucleate female gametophyte. (B) Higher magnifi cation view of tissue within the red box in (A). Three of the four cells of the female gametophyte can be seen (one of the two synergids is not visible in this section). Small arrows point to a few of the many small starch grains in the nucellus. (C) Median longitudinal section of the nucellus of an ovule stained with toluidine blue and observed with polarized light. Raphides can be seen in several of the cells which are otherwise highly vacuolate. Abbreviations : ccn, central cell nucleus; ch, chalaza; egg, egg cell; fg, female gametophyte; fun, funicle; ii, inner integument; nuc, nucellus; oi, outer integument; r, raphide; syn, synergid cell; zm, zone of megasporogenesis. Bars = 200 μ m (A), 50 μ m (B), and 10 μ m (C).

909May 2013] FRIEDMAN AND BACHELIER―EMBRYO-NOURISHING STRATEGIES IN SEEDS OF EARLY ANGIOSPERMS

only be observed prior to application of these stains or after staining protocols that do not involve acids (e.g., toluidine blue) ( Fig. 3B ).

Early seed development in Trimenia moorei — Pollen tube discharge was observed in the synergids of several mature fe-male gametophytes, but double fertilization of the haploid egg cell and haploid central cell (syngamy) was not captured in any of the histological preparations, as is usually the case. After fertilization, a few additional small starch grains are formed in the epidermal cells of the nucellus, in the zygote, and around the diploid primary endosperm nucleus. The endosperm ex-tends for the entire length of the nucellus but the fi rst divisions were not observed at the chalazal end. Successive transverse cell divisions in the large micropylar chamber create a uniseri-ate endosperm ( Fig. 4A , see also Fig. 8C ).

Further cell divisions of the endosperm around the undivided zygote yield smaller and more densely cytoplasmic cells ( Fig. 4B , see also Fig. 8D ). Only after the completion of this fi rst phase of cellular endosperm development does the zygote undergo its fi rst mitotic and cytokinetic division ( Fig. 5 ). With the excep-tion of the very apex and epidermis, most of the nucellus re-mains devoid of stored nutrients ( Fig. 5B, C ). During this period of early endosperm development, raphides continue to accumulate in nucellar cells.

Seed maturation in Trimenia moorei and T. neocaledonica — Initial development of the embryo proceeds at a much slower rate than the endosperm ( Figs. 5, 6 ). By the time the embryo is globular, the endosperm has begun to expand radially and the immediately surrounding cells of the nucellus are beginning to

polarized microscopy, as well as digital imaging. Fluorescence of Auramine-O was visualized with an HBO 100W burner with excitation fi lter (BP-530-585 nm), dichroic mirror (FT600), and barrier fi lter (LP615) (Zeiss). Pictures, line draw-ings, and fi gures were all processed and edited using Adobe Creative Suite 5 (Adobe Systems, San Jose, California, USA). Image manipulations were re-stricted to operations that were applied to the entire image, except as noted in specifi c fi gure legends. Boxes outlined in solid black lines in a micrograph in-dicate a digital inset from an adjacent section of the same series of histological sections. Insets outlined with a solid red line are linked to a higher magnifi ca-tion image of the boxed portion of a section. All ovules and seeds are oriented with the apex of the nucellus upwards ( Figs. 2-8 ).

RESULTS

Ovule development prior to fertilization in Trimenia moorei — The ovule of T. moorei is crassinucellate ( sensu Endress, 2011 ), anatropous (syntropous), and bitegmic. Both the annular inner integument and the thicker hood-shaped outer integument are involved in the formation of the micropyle. Starch is accumulated prior to fertilization in the center of the massive nucellus tissue, but it is almost entirely consumed during megasporogenesis and female gametophyte develop-ment ( Fig. 2 , see also Fig. 8A ; for detail see Figs. 1, 2, and 3 in Bachelier and Friedman, 2011 ).

At fertilization, there are only a few small starch grains remaining around the tip of the dominant mature female game-tophyte (more than one female gametophyte can develop in each individual ovule). Most cells of the nucellus are devoid of starch and highly vacuolated ( Fig. 3A , see also Fig. 8B ). Polar-ized light reveals, however, that many nucellus cells start to accumulate raphides ( Fig. 3B ). These crystals, however, are dissolved by the acids during PAS and ABB staining, and can

Fig. 4. Immature seeds of Trimenia moorei with uniseriate endosperm and undivided zygote. Median longitudinal sections of young (A) and later postfertilization stages (B) stained with PAS and toluidine blue. (A) Early uniseriate endosperm. (B) Later uniseriate endosperm with small cells surround-ing the zygote. Large arrowheads point to micropylar half of the endosperm in (A). Small arrows highlight a few of the starch grains present at the apex of the nucellus. Abbreviations : ch, chalaza; end, endosperm; ii, inner integument; nuc, nucellus; oi, outer integument; zyg, zygote. Bars = 200 μ m (A), 50 μ m (inset in A, B).

910 AMERICAN JOURNAL OF BOTANY [Vol. 100

Nutrient storage in mature seeds of Trimenia moorei — Large starch grains are found in the cells of the embryo suspensor, the protoderm, and the cotyledon primordia ( Fig. 7C ). The en-dosperm contains small starch grains at its periphery and larger grains in the cells at the center of the tissue (in line with the axis of the embryo). In addition to starch, the endosperm contains signifi cant quantities of protein bodies and lipid droplets ( Fig. 7A, D, E ). Clusters of raphides (from nucellus cells that have been crushed during the expansion of the en-dosperm) are present between the endosperm and the persis-tent layer of nucellus tissue ( Fig. 7F ). The persistent layer of the nucellus, however, remains devoid of stored nutrients at seed maturity/dormancy ( Fig. 7D, E ). Thus, there is no evi-dence for a perisperm in T. moorei . Although we were unable to obtain mature seeds of T. neocaledonica , our developmen-tal analysis indicates there is no evidence for the sequestration of embryo-nourishing reserves in the nucellus in this species either.

DISCUSSION

Seed development and structure in Trimenia moorei — In T . moorei , previous studies reported that the majority of the tissue charged with storing and contributing nutrients to the embryo in mature seeds was the nucellus, functioning as a perisperm ( Prakash, 1998 ; Yamada et al., 2003 ). In addition, these reports

collapse ( Fig. 6 ). At this stage and later, starch grains are still found in the embryo, the epidermal layer of the nucellus, and the tip of the nucellus, but nowhere else.

As the seed develops to maturity, cell divisions within the endosperm are associated with its continued radial expansion ( Fig. 6 ). During this period of development, the nucellus be-comes progressively more diffi cult to observe in histological sections of both species ( Figs. 5, 6 ). The accumulation of raphi-des may facilitate the rupture and collapse of nucellar cells as the radial expansion of the endosperm crushes the remaining nucellar cells ( Fig. 6D ). Nevertheless, it is possible to reconstruct all of the basic developmental events associated with seed mat-uration in Trimenia (see Fig. 8E ).

The complete radial expansion of the endosperm fi lls nearly the entirety of the inner mature seed, but does not entirely obliter-ate the nucellar tissue of the ovule. The nucellus is reduced to a few cell layers at the chalazal end of the seed, a single epidermal cell layer further up, and is entirely crushed only around the apex ( Fig. 7A , see also Fig. 8F ). Continued growth and development of the embryo lead to the consumption of the immediately proxi-mate endosperm tissue. Thus, the mature seed is largely fi lled with an endosperm surrounded by a very thin persistent layer of nucellus ( Fig. 7A , see also Fig. 8F ). The embryo remains rela-tively small but is well-differentiated, with a multicellular and multiseriate suspensor, a protoderm and procambial tissue. Two cotyledon primordia are minute but present ( Fig. 7B, C ).

Fig. 5. Immature seeds of Trimenia moorei with multiseriate endosperm and two-celled and three-celled proembryos. Median longitudinal sections stained with PAS and toluidine blue. (A) Seed with three-celled embryo. Large arrowheads identify endosperm. Black boxes indicate digital insets from adjacent histological sections to show the full longitudinal extent of the endosperm. (B) Higher magnifi cation view of tissue within the red box in (A). The nucellus is largely devoid of any storage compounds although a few small starch grains are evident in the nucellar epidermis (small arrows). (C) Two-celled embryo surrounded by a thin layer of endosperm. Small arrows identify a few of the starch grains present at the micropylar end and epidermal cells of the nucellus. Abbreviations : ch, chalaza; emb, embryo; end, endosperm; fun, funicle; ii, inner integument; nuc, nucellus; oi, outer integument. Bars = 200 μ m (A), 100 μ m (B), 50 μ m (C).

911May 2013] FRIEDMAN AND BACHELIER―EMBRYO-NOURISHING STRATEGIES IN SEEDS OF EARLY ANGIOSPERMS

Thus, the fi ndings of Prakash (1998) and Yamada et al. (2003 , 2008 ), who concluded that the majority of the embryo-nourishing tissue within the mature seeds of Trimenia moorei is a perisperm (derived from the nucellus), cannot be sustained. To some extent, nutrients are stored in the small but well dif-ferentiated dicotyledonous embryo itself, but by the time of seed dormancy, the main nutrient-storing and embryo-nourishing tissue in seeds of T . moorei is a genetically biparental diploid endosperm.

Endosperm development in Trimenia and Austrobaileyales — In Trimenia , endosperm is ab initio cellular ( Prakash, 1998 ). This pattern of endosperm development appears to be general to all other members of the Austrobaileyales, including Aus-trobaileya ( Endress, 1980 ), Illicium ( Hayashi, 1963a ; Floyd and Friedman, 2000 , 2001 ), Kadsura ( Hayashi, 1963b ), and Schisandra ( Hayashi, 1963b ; Kapil and Jalan, 1964 ). Endosperm development begins with a division of the primary endosperm cell that yields two large cells in T . moorei ( Prakash, 1998 ), and a large micropylar and a smaller chalazal cell in Illicium ( Floyd

indicated that the endosperm was minute and did not play a primary role in nourishing the embryo during seed develop-ment. A perisperm smaller than the endosperm was later re-ported in fossil seeds of Trimeniaceae, and the presence of a copious perisperm in extant Trimeniaceae was interpreted as a derived character that evolved secondarily within the family ( Yamada et al., 2008 ).

Our re-evaluation of the development of seeds in Trimenia moorei clearly shows that the previous report of a vermiform endosperm extending for the entire length of the nucellus, but occupying only a small central portion of the seed, was proba-bly based on an immature stage of seed development (compare Figs. 4 and 5A in Prakash, 1998 with Fig. 8C, D in this study). Our developmental work on Trimenia also shows that while the nucellus is substantial and contains signifi cant amounts of starch prior to fertilization, it does not play any role in nutrient storage and embryo-nourishing during seed development. Rather, the nucellus remains largely devoid of stored nutrients from the time of fertilization through seed maturation and is almost entirely obliterated by the expansion of the endosperm.

Fig. 6. Immature fruits and seeds of Trimenia neocaledonica with large multiseriate endosperms and multicellular embryos. Median longitudinal sec-tions of younger (A, B) and older stages (C, D, E) stained with toluidine blue. Large arrowheads point to endosperm. (A) Endosperm is multiseriate, but has only just begun to expand radially. (B) Higher magnifi cation view of tissue within the red box in (A) showing the embryo and immediately surrounding endosperm. (C) Endosperm has continued to expand radially. Black boxes indicate digital insets from adjacent histological sections to show the full longi-tudinal extent of the endosperm. (D) Higher magnifi cation view of tissue within the red box in (C) showing a cluster of raphides in the nucellus. (E) Late stage of endosperm development with multicellular embryo. At this point, inner portions of the nucellus have been crushed by the radial expansion of the endosperm. Abbreviations : ch, chalaza; emb, embryo; end, endosperm; fun, funicle; ii, inner integument; nuc, nucellus; oi, outer integument; r, raphide. Bars = 500 μ m (A, C), 100 μ m (E), 20 μ m (B, D).

912 AMERICAN JOURNAL OF BOTANY [Vol. 100

present in the mature endosperm of Trimenia moorei . Although most of the earlier studies of seed development in members of the Austrobaileyales did not undertake extensive histological analyses of endosperm contents, in Illicium , the endosperm contains proteins and lipids, but starch is reported to be absent ( Floyd and Friedman, 2001 ). In Schisandra , both starch and oils are present in the mature endosperm but it is unknown whether proteins are accumulated ( Kapil and Jalan, 1964 ). In Austrobaileya , starch has been reported in the large ruminate endosperm, although tests for lipids and proteins were not per-formed ( Endress, 1980 ). Thus, there may be a modest amount of variation in the nature and relative proportion of the differ-ent types of storage compounds among genera of Austrobai-leyales. Given the different functional and nutritional roles of starch, proteins, and lipids in seeds ( Kitajima and Myers, 2008 ; Soriano et al., 2011 ), it would be interesting to examine how the different proportions of these storage reserves in the endosperms of the diverse members of the Austrobaileyales might correlate with patterns of seedling germination and establishment.

and Friedman, 2000 , 2001 ), Kadsura ( Hayashi, 1963b ) and Schisandra ( Hayashi, 1963b ; Kapil and Jalan, 1964 ). Also, both Trimenia and Illicium share a uniseriate developmental elabo-ration of the micropylar cell of the two-celled endosperm ( Floyd and Friedman, 2000 , 2001 ; this study).

The formation and differentiation of a genetically biparen-tal and diploid endosperm appears to be fundamentally similar in all members of the Austrobaileyales studied to date. Even though the early stages of endosperm development and patterns of cell divisions remain unstudied in a few members of the Aus-trobaileyales (particularly Austrobaileya ), endosperm in all Austrobaileyales studied to date develops into a large nutrient-storing and embryo-provisioning tissue. This tissue surrounds a small but well-differentiated dicotyledonous embryo and fi lls most of the seed ( Trimenia , this study; Schisandra , Hayashi, 1963b ; Kapil and Jalan, 1964 ; Chien et al., 2011 ; Kadsura , Hayashi, 1963b ; Austrobaileya , Endress, 1980 ; Illicium , Hayashi, 1963a ; Floyd and Friedman, 2000 , 2001 ).

In the current study, we clearly show that a small amount of starch and signifi cant quantities of protein bodies and lipids are

Fig. 7. Mature seed and embryo of Trimenia moorei . Median longitudinal sections stained with PAS (A, B), PAS and aniline blue black (D), Auramine-O (E), or toluidine blue (F). Large black arrowheads in D and large white arrowheads in E point to contact zone between endosperm and persistent nucellus tissue. Small black arrows in B identify starch grains and small white arrows in E indicate lipids. Asterisks mark cotyledons. (A) Seed with a small dicoty-ledonous embryo and a full-fl edged endosperm surrounded by a persistent layer of nucellus tissue. (B) Higher magnifi cation view of tissue within the upper red box in (A) of the embryo with differentiated protoderm and procambial strand, and cotyledonary primordia crushing the surrounding endosperm. (C) Whole embryo with two cotyledon primordia (asterisks) and adherent endosperm, dissected from a seed. (D-F) Contact zone between endosperm and the persistent layer of nucellus. Each of these panels is taken from the general area denoted by the lower red box in (A). (D) Protein bodies in the endosperm but not in the nucellus layer. (E) Lipid droplets in the endosperm (indicated by small white arrows) but not in the nucellus layer. (F) Raphides (indicated by large white arrowheads) between the endosperm and the persistent nucellus epidermal layer visualized with crossed polarizing fi lters. Abbreviations : emb, embryo; end, endosperm; nuc, nucellus; s, suspensor. Bars = 500 μ m (A), 200 μ m (B, C), 20 μ m (D-F).

913May 2013] FRIEDMAN AND BACHELIER―EMBRYO-NOURISHING STRATEGIES IN SEEDS OF EARLY ANGIOSPERMS

Modest amounts of starch are also accumulated in the nucellus of some Calycanthaceae (Laurales; Mathur, 1968 ), Annonaceae (Magnoliales; Lora et al., 2010 ), and Piperaceae (Piperales; Lei et al., 2002 ) prior to fertilization. In Hydatellaceae and Piper-aceae, the nucellus persists after fertilization and differentiates into the main nutrient storage and embryo-nourishing tissue (perisperm) in the seed. In Calycanthaceae ( Mathur, 1968 ) and Annonaceae ( Lora et al., 2010 ), the nucellus does not accumu-late signifi cant reserves after fertilization and the endosperm is the primary embryo-nourishing tissue within the seed. For now, the developmental and evolutionary history of storage com-pounds in nucellar tissues during the early diversifi cation of angiosperms remains opaque.

Conclusions — More than a century after the discovery that endosperm is formed as a consequence of a second fertiliza-tion event in angiosperms, the evolutionary and developmental transition from seeds with a maternally derived haploid nutrient-storing and embryo-nourishing tissue (the female gameto-phyte of gymnosperms) to seeds with a biparental full-fl edged endosperm (most angiosperms) remains poorly understood. Among early angiosperm lineages, endosperm provisions for

Storage compounds in the nucellus in early diverging angio-sperms — Given the complete absence of a perisperm in the ma-ture seeds of extant Trimenia and all other members of the Austrobaileyales studied to date (this study; Hayashi, 1963a , 1963b ; Kapil and Jalan, 1964 ; Endress, 1980 ; Chien et al., 2011 ; Floyd and Friedman, 2000 , 2001 ), the presence of a nutrient-storing and embryo-nourishing biparental endosperm remains the most parsimonious reconstruction of the ancestral condition for angiosperms as a whole.

However, the accumulation of starch prior to fertilization in the ovule of Trimenia moorei ( Bachelier and Friedman, 2011 ) raises the question of whether this might represent an ancient develop-mental feature of the nucellus of angiosperms. In Amborella , there is no evidence for the accumulation of any signifi cant storage com-pounds in the nucellus prior to or after fertilization ( Tobe et al., 2000 ; Floyd and Friedman, 2001 ; Friedman, 2006 ). Among other early divergent lineages of fl owering plants, the accumulation of starch in the nucellus prior to fertilization has been reported in Hydatellaceae (Nymphaeales; Friedman, 2008 ; Rudall et al., 2008 ), but not in members of the Nymphaeaceae and Cabom-baceae ( Seaton, 1908 ; Khanna, 1967 ; Schneider, 1978 ; Van Miegroet and Dujardin, 1992 ).

Fig. 8. Endosperm development in Trimenia . Line drawings of ideal median longitudinal section of ovules and seeds, outlined with a solid black line. Biparental diploid endosperm shown in blue, embryo in gray, and maternal nucellus tissue in green . (A) Young ovule during megasporogenesis (black dots represent starch grains; yellow solid ovals represent megaspore mother cells and tetrads). (B) Ovule with mature female gametophyte in yellow. (C) Seed with uniseriate endosperm and undivided zygote. The nucellus is still prominent but does not contain any seed storage compounds. (D) Seed with multiseri-ate endosperm and 2-celled proembryo and nucellus that still occupies a signifi cant volume. (E) Seed with multiseriate endosperm and multicellular em-bryo. (F) Seed at maturity/dormancy, with copious full-fl edged endosperm , dicotyledonous embryo, and vestiges of the nucellus that has been obliterated by the expansion of the endosperm.

914 AMERICAN JOURNAL OF BOTANY [Vol. 100

FRIEDMAN , W. E. , AND J. H. WILLIAMS . 2004 . Developmental evolution of the sexual process in ancient fl owering plants lineages. Plant Cell 16 : S119 – S132 .

HAYASHI , Y . 1963a . The embryology of the family Magnoliaceae sens. lat. I. Megasporogenesis, female gametophyte and embryogeny of Illicium anisatum L . Science Reports of the Tôhoku Imperial University, ser. 4 (Biol.) 29 : 27 – 33 .

HAYASHI , Y . 1963b . The embryology of the family Magnoliaceae sens. lat. II. Megasporogenesis, female gametophyte and embryology of Schisandra repanda Radlkofer and Kadsura japonica Dunal . Science Reports of the Tôhoku Imperial University, ser. 4 (Biol.) 29 : 403 – 411 .

KAPIL , R. N. , AND S. JALAN . 1964 . Schisandra Michaux—its embryology and systematic position. Botaniska Notiser 117 : 285 – 306 .

KHANNA , P. 1967 . Morphological and embryological studies in Nympha-eaceae. III. Victoria cruziana D’Orb. and Nymphaea stellata Willd. Botanical Magazine 80 : 305 – 312 .

KITAJIMA , K. , AND J. A. MYERS . 2008 . Seedling ecophysiology: Strategies towards achievement of positive carbon balance. In M. A. Leck, V. T. Parker, and R. L. Simpson [eds], Seedling ecology and evolution, 172–188. Cambridge University Press, Cambridge, UK.

LEI , L.-G. , Z. Y. WU , AND H. X. LIANG . 2002 . Embryology of Zippelia begoniaefolia (Piperaceae) and its systematic relationships. Botanical Journal of the Linnean Society. Linnean Society of London 140 : 49 – 64 .

LORA , J. , J. I. HORMAZA , AND M. HERRERO . 2010 . The progamic phase of an early-divergent angiosperm, Annona cherimola (Annonaceae). Annals of Botany 105 : 221 – 231 .

MATHEWS , S. , AND M. J. DONOGHUE . 1999 . The root of angiosperm phylogeny inferred from duplicate phytochrome genes. Science 286 : 947 – 950 .

MATHUR , S. L. 1968 . Development of the male and female gametophytes of Calycanthus fertilis Walt. Proceedings of the National Institute of Science, India , B, Biological Sciences 34: 323–329.

PARKINSON , C. L. , K. L. ADAMS , AND J. D. PALMER . 1999 . Multigene anal-yses identify the three earliest lineages of extant fl owering plants. Current Biology 9 : 1485 – 1488 .

PRAKASH , N. 1998 . An embryological study of Piptocalyx moorei (Trimeniaceae). In B. Bhatia, A. K. Shukla, and H. L. Sharma [eds.], Plant form and function, 207–216. Angkor, New Delhi, India.

QIU , Y.-L. , J. LEE , F. BERNASCONI-QUADRONI , D. E. SOLTIS , P. S. SOLTIS , M. ZANIS , E. A. ZIMMER , ET AL . 1999 . The earliest angiosperms: Evidence from mitochondrial, plastid and nuclear genomes. Nature 402 : 404 – 407 .

RUDALL , P. J. 2006 . How many nuclei make an embryo sac in fl owering plants? BioEssays 28 : 1067 – 1071 .

RUDALL , P. J. , T. ELDRIDGE , J. TRATT , M. M. RAMSAY , R. E. TUCKETT , S. Y. SMITH , M. E. COLLINSON , ET AL . 2009 . Seed fertilization, develop-ment, and germination in Hydatellaceae (Nymphaeales): Implications for endosperm evolution in early angiosperms. American Journal of Botany 96 : 1581 – 1593 .

RUDALL , P. J. , M. V. REMIZOWA , A. S. BEER , E. BRADSHAW , D. W. STEVENSON , T. D. MACFARLANE , R. E. TUCKETT , ET AL . 2008 . Comparative ovule and megagametophyte development in Hydatellaceae and water lilies reveal a mosaic of features among the earliest angiosperms. Annals of Botany 101 : 941 – 956 .

SAARELA , J. M. , H. S. RAI , J. A. DOYLE , P. K. ENDRESS , S. MATHEWS , A. D. MARCHANT , B. G. BRIGGS , AND S. W. GRAHAM . 2007 . Hydatellaceae identifi ed as a new branch near the base of the angiosperm phyloge-netic tree. Nature 446 : 312 – 315 .

SCHNEIDER , E. L. 1978 . Morphological studies of the Nymphaeaceae. IX. The seed of Barclaya longifolia Wall. Botanical Gazette (Chicago, Illinois) 139 : 223 – 230 .

SEATON , S. 1908 . The development of the embryo–sac of Nymphaea ad-vena. Bulletin of the Torrey Botanical Club 35 : 283 – 290 .

SOLTIS , P. S. , D. E. SOLTIS , AND M. W. CHASE . 1999 . Angiosperm phylog-eny inferred from multiple genes as a tool for comparative biology. Nature 402 : 402 – 404 .

SORIANO , D. , A. OROZCO-SEGOVIA , J. MÁRQUEZ-GUZMÁN , K. KITAJIMA , A. GAMBOA-DE BUEN , AND P. HUANTE . 2011 . Seed reserve composition

and nourishes the embryo in Amborella and members of the Austrobaileyales. In the case of Amborella , the endosperm is triploid, while in Austrobaileyales, it is diploid. As fi rst sug-gested by Williams and Friedman (2002) , current evidence points to a diploid condition for endosperm as being plesiomor-phic for angiosperms as a whole (see Friedman et al., 2012 ).

In Nymphaeales, however, the diploid endosperm is minute, and the entirety of nutrient storage and embryo-nourishing within the seed is associated with the nucellus and its formation into a perisperm. Previous reports of a minute endosperm coupled with a perisperm in the seeds of Trimenia moorei ( Prakash, 1998 ; Yamada et al., 2003 , 2008 ) raised the possibility that the common ancestors of the Nymphaeales and Austrobaileyales could both have used a perisperm as the primary embryo-nourishing tissue within the seed, but our current fi ndings for Trimenia diminish the likelihood that the common ancestor of Austrobaileyales formed an embryo-nourishing perisperm. In either case, the de-velopmental and functional biology of the diploid endosperm of Trimenia (and other Austrobaileyales) differs markedly from the diploid endosperms of Nymphaeales, and is fundamentally simi-lar to the triploid endosperms of most other angiosperms.

LITERATURE CITED

BACHELIER , J. B. , AND W. E. FRIEDMAN . 2011 . Female gamete competi-tion in an ancient angiosperm lineage. Proceedings of the National Academy of Sciences, USA 108 : 12360 – 12365 .

CHIEN , C.-T. , S.-Y. CHEN , J. M. BASKIN , AND C. C. BASKIN . 2011 . Morphophysiological dormancy in seeds of the ANA grade an-giosperm Schisandra arisanensis (Schisandraceae). Plant Species Biology 26 : 99 – 104 .

ENDRESS , P. K. 1980 . The reproductive structures and systematic posi-tion of the Austrobaileyaceae. Botanische Jahrbücher für Systematik, Pfl anzengeschichte und Pfl anzengeographie 101 : 393 – 433 .

ENDRESS , P. K. 2011 . Angiosperm ovules: Diversity, development, evolu-tion. Annals of Botany 107 : 1465 – 1489 .

FLOYD , S. K. , AND W. E. FRIEDMAN . 2000 . Evolution of endosperm de-velopmental patterns among basal fl owering plants. International Journal of Plant Sciences 161 : S57 – S81 .

FLOYD , S. K. , AND W. E. FRIEDMAN . 2001 . Developmental evolution of endosperm in basal angiosperms: Evidence from Amborella (Amborellaceae), Nuphar (Nymphaeaceae), and Illicium (Illiciaceae). Plant Systematics and Evolution 228 : 153 – 169 .

FRIEDMAN , W. E. 2006 . Embryological evidence for developmental labil-ity during early angiosperm evolution. Nature 441 : 337 – 340 .

FRIEDMAN , W. E. 2008 . Hydatellaceae are water lilies with gymnosper-mous tendencies. Nature 453 : 94 – 97 .

FRIEDMAN , W. E. , J. B. BACHELIER , AND J. I. HORMAZA . 2012 . Embryology in Trithuria submersa (Hydatellaceae) and relationships between em-bryo, endosperm, and perisperm in early-diverging fl owering plants. American Journal of Botany 99 : 1083 – 1095 .

FRIEDMAN , W. E. , W. N. GALLUP , AND J. H. WILLIAMS . 2003 . Gametophyte development in Kadsura : Implications for Schisandraceae, Austro-baileyales, and the early evolution of fl owering plants. International Journal of Plant Sciences 164 : S293 – S305 .

FRIEDMAN , W. E. , E. N. MADRID , AND J. H. WILLIAMS . 2008 . Origin of the fi ttest and survival of the fi ttest: Relating female gametophyte development to endosperm genetics. International Journal of Plant Sciences 169 : 79 – 92 .

FRIEDMAN , W. E. , AND K. C. RYERSON . 2009 . Reconstructing the ances-tral female gametophyte of angiosperms: Insights from Amborella and other ancient lineages of fl owering plants. American Journal of Botany 96 : 129 – 143 .

FRIEDMAN , W. E. , AND J. H. WILLIAMS . 2003 . Modularity in the angio-sperm female gametophyte and its bearing on the early evolution of endosperm in fl owering plants. Evolution; International Journal of Organic Evolution 57 : 216 – 230 .

915May 2013] FRIEDMAN AND BACHELIER―EMBRYO-NOURISHING STRATEGIES IN SEEDS OF EARLY ANGIOSPERMS

in 19 tree species of a tropical deciduous forest in Mexico and its re-lationship to seed germination and seedling growth. Annals of Botany 107 : 939 – 951 .

STEVENS , P. F. 2001 onward . Angiosperm phylogeny website, version 9, June 2008 [more or less continuously updated]. http://www.mobot.org/MOBOT/research/APweb/ . [accessed March 2012].

TOBE , H. , T. JAFFRÉ , AND P. H. RAVEN . 2000 . Embryology of Amborella (Amborellaceae): descriptions and polarity of character states. Journal of Plant Research 113 : 271 – 280 .

TOBE , H. , Y. KIMOTO , AND N. PRAKASH . 2007 . Development and structure of the female gametophyte in Austrobaileya scandens (Austrobaileyaceae). Journal of Plant Research 120 : 431 – 436 .

VAN MIEGROET , F. , AND M. DUJARDIN . 1992 . Cytologie et histologie de la reproduction chez le Nymphaea heudelotii. Canadian Journal of Botany 70 : 1991 – 1996 .

WILLIAMS , J. H. 2008 . Novelties of the fl owering plant pollen tube un-derlie diversifi cation of a key life history stage. Proceedings of the National Academy of Sciences, USA 105 : 11259 – 11263 .

WILLIAMS , J. H. , AND W. E. FRIEDMAN . 2002 . Identifi cation of diploid en-dosperm in an early angiosperm lineage. Nature 415 : 522 – 526 .

WILLIAMS , J. H. , AND W. E. FRIEDMAN . 2004 . The four–celled female gametophyte of Illicium (Illiciaceae; Austrobaileyales): Implications for understanding the origin and early evolution of monocots, eumag-noliids, and eudicots. American Journal of Botany 91 : 332 – 351 .

YAMADA , T. , R. IMAICHI , N. PRAKASH , AND M. KATO . 2003 . Developmental morphology of the ovules and seeds of Austrobaileyales. Australian Journal of Botany 51 : 555 – 564 .

YAMADA , T. , H. NISHIDA , M. UMEBAYASHI , K. UEMURA , AND M. KATO . 2008 . Oldest record of Trimeniaceae from the Early Cretaceous of northern Japan. BMC Evolutionary Biology 8 : 135 .