Prolonged embryogenesis in Austrobaileya scandens (Austrobaileyaceae): its ecological and evolutionary significance

Plant material collection

The majority of reproductive material of A. scandens C. T. White was collected in the field in Queensland, Australia (2007 and 2012). Fruits of different sizes (developmental stages) were collected in the canopies of supporting trees and kept in humid bags until transferred to the laboratories of the Commonwealth Scientific and Industrial Research Organisation (CSIRO) in Atherton (Australia). There, their maximum length was measured and the fresh weight was recorded with a precision scale before fixation. Additionally, flowers and fruits of A. scandens were collected at different developmental stages from cultivated plants at the University of Colorado, Boulder, CO, USA (2010) and from the Botanical Garden of the University of Zurich, Zurich, Switzerland, by P. K. Endress (2010).

Fresh mature seeds were also collected in the field after fruit abscission using a net at the base of the liana. These fresh seeds were sowed ex situ in pots with high porosity PRO-MIX (Premier Tech Horticulture and Agriculture Group, Rivière-du-Loup, QC, Canada) growing medium. Pots were kept at warm temperatures (25–35°C) and high humidity (60–80%) for 1 yr, and watered regularly to maintain a saturated soil moisture. Seeds were examined every 2 months until radicle emergence, and after germination, embryos were collected at different developmental stages based on radicle length.

Material fixation

Plant material was fixed with either 4% acrolein (Polysciences Inc., Warrington, PA, USA) in a modified piperazine-N,N′-bis(2-ethanesulfonic acid) (PIPES) buffer adjusted to pH 6.8 (50 mM PIPES and 1 mM MgSO₄ from BDH, London, UK; and 5 mM EGTA from Research Organics Inc., Cleveland, OH, USA), formalin acetic acid (FAA) (Johansen, 1940), or 4% paraformaldehyde (Solís et al., 2008) in 0.1 M phosphate-buffered saline. All fixed specimens were rinsed in their respective buffers (PIPES or PBS), and dehydrated through a graded ethanol series up to 70% ethanol.

Microscopy

Ovules and seeds were dehydrated with an ethanol series up to 100% ethanol, infiltrated with and embedded in glycol methacrylate resin (JB-4 Polysciences, Warrington, PA, USA) or Kulzer Technovit 8100 (Electron Microscopy Sciences, Hatfield, PA, USA), and sectioned with a rotary microtome (Microm HM360 from Thermo Fisher Scientific, Waltham, MA, USA). While ovules and young (smaller) seeds were sectioned with glass knives into 4-μm-thick ribbons, larger specimens were sectioned at 6–10 μm thickness with coated steel knives. Sections were mounted onto slides, stained with a periodic acid–Schiff's reaction (PAS) to detect insoluble carbohydrates (Feder & O'Brien, 1968), and counterstained with aniline blue–black (ABB) in 7% acetic acid to detect proteins (Linskens & Esser, 1957). A solution of 0.01% Auramine-O in 0.05 M Tris/HCl buffer, pH 7.2, was used to detect lipids (Hughes & McCully, 1975).

Digital imaging

Photographs of flowers, fruits, and seedlings were taken in the field using a Canon Power S50 digital camera. Images of embryos dissected from germinating seeds were taken using a Discovery AxioVision dissecting microscope (Carl Zeiss, Oberkochen, Germany). Photographs of microtome sections, combining bright field with differential interference contrast, were taken using the Zeiss Axio Imager Z2 microscope equipped with Zeiss High Resolution AxioCam digital cameras (Carl Zeiss). Fluorescence of auramine-O was visualized with an HBO 100W burner with excitation filter (BP-530–585 nm), dichroic mirror (FT600), and barrier filter (LP615) (Carl Zeiss). All figures were edited using Adobe Creative Suite 5 (Adobe Systems, San Jose, CA, USA). In Figs 1-9, all panels are arranged in chronological sequences from the youngest stage of development (left or upper) to the oldest (right or bottom).

Anthesis

Floral buds and anthetic flowers co-occur on the same plant with mature fruits from the previous year, indicating that at least 12 months must elapse from anthesis to fruit maturation in A. scandens (Fig. 1). At anthesis, the flowers are strongly (unpleasantly) scented, fly-pollinated, and protogynous. Initially, the perianth forms a narrow opening and only the stigmas of the apocarpous gynoecium, which are postgenitally united by secretion, are visible in the center of the flower (Fig. 1a; see also Endress, 1980, 2008). Over the following 2 d, each flower enters the male phase and the perianth opens more widely. The laminar stamens become reflexed and exposed, whereas the carpels are hidden by (inner) staminodia (Fig. 1b). Five days after hand pollination, the perianth, stamens, and staminodes abscise and only the fertilized carpels remain (Fig. 1c).

Early seed development

At anthesis, the mature monosporic female gametophyte (formed by the chalazal-most megaspore of a triad of megaspores) is four-celled and four-nucleate (see also Tobe et al., 2007; Supporting Information, Fig. S1), with an egg cell, two small synergids, and a single uninucleate (haploid) central cell (Fig. 2a). After double fertilization of the female gametophyte, one synergid persists (data not shown) along with the zygote at the micropylar end.

The (genetically biparental) diploid primary endosperm nucleus migrates towards the chalazal pole. The zygote remains undivided while the primary endosperm nucleus undergoes a first cell division to yield a long micropylar endosperm cell and a much smaller chalazal endosperm cell (Fig. 2b). Like the zygote, the micropylar endosperm cell initially remains undivided while the chalazal cell initiates a number of cell divisions in multiple planes to produce roughly a dozen cells (Fig. 2c). The micropylar endosperm cell then initiates a series of uniseriate transverse divisions (Fig. 2d). One month after fertilization, the micropylar endosperm (derived from the micropylar cell of the two-celled endosperm, sensu Floyd & Friedman, 2000) has become multiseriate and tiered, and can be distinguished from the chalazal endosperm domain derived from the chalazal cell of the two-celled endosperm (Fig. 3a).

At anthesis, the nucellus is devoid of starch (Fig. 2e). During early seed development, however, a noticeable accumulation of insoluble polysaccharide reserves (starch grains) begins at the chalazal end of the nucellus, just below the chalazal endosperm cell (Fig. 2f). Later, a modest number of conspicuous starch grains are observed in all of the cells of the persistent maternal tissue of the nucellus, whereas the quiescent zygote and expanding endosperm remain devoid of any reserves (Fig. 2g,h).

The zygote remains quiescent (Fig. 3b) until c. 2 months after fertilization (Fig. 3d), when it undergoes a first cell division (Fig. 3e). At this time, the endosperm starts to expand radially. Cells of the nucellus are highly vacuolate (Fig. 3c) and nucellar starch reserves have been removed. From this point in seed development, expansion of the endosperm progressively crushes the surrounding maternal tissues of the nucellus (Fig. 3f). Eventually, the nucellus is entirely obliterated by the expanding endosperm (Fig. 4).

Late seed development

Approximately 5 months after fertilization, the proliferating endosperm becomes distinctly ruminate (Fig. 4a), and the nucellus has been entirely obliterated. The embryo proper is globular (Fig. 4b) and contains starch (Fig. 4c). Between 5 and 12 months after fertilization, seeds roughly double in size, and at maturity, the endosperm occupies the vast majority of the volume (Fig. 4d). During the final 6 months of seed development on the mother plant, significant amounts of starch accumulate within cells of the endosperm (Fig. 4e), and at seed maturity (fruit abscission), both the endosperm and the embryo are filled with copious starch grains (Fig. 4f,g). Proteins and lipids also accumulate in the endosperm (Figs 4h, 7a,b), but to a lesser extent in the embryo (data not shown).

During the 12 months after fertilization, fruits increased in length (Fig. 1d,e) up to 7.0 cm with a fresh weight of up to 40 g (Fig. 1f). The green pericarp and mesocarp then turned an intense orange color, and before abscission, each fruit became significantly lighter, with weights of c. 26 g (Fig. 1g). The average number of seeds per fruit was 6.0 (range 4–11; n = 20), and the individual fresh weight per seed was just over 3 g (n = 50) (Fig. 1h). When the fruits abscised, the dicotyledonous embryo was minute (0.6% of the total seed volume) but morphologically well differentiated, with a root−shoot axis, short hypocotyl, and two cotyledons (Fig. 4e,f).

Post-dispersal seed maturation, germination, and seedling establishment

In seeds that have been dispersed from the mother plant, the minute embryo does not initially enlarge significantly (Fig. 5a,b). With time, the cotyledons begin to expand (Fig. 5d,f), and c. 12 months after dispersal (24 months after fertilization) have almost entirely consumed and displaced the endosperm (Fig. 5h).

Before radicle emergence, as early as 6 months after sowing (18 months after fertilization) (Fig. 6a), starch grains disappear from the embryo while vascular tissues differentiate (Fig. 6b) in the cotyledons and root−shoot axis. Proteins, starch, and lipids, although still abundant in the endosperm, start to break down before radicle emergence (Figs 6c, 7c). After radicle emergence (Fig. 6d), proteins and starch are withdrawn from the endosperm (Fig. 6f,i) and are putatively absorbed by the expanding cotyledons, which stain intensely for proteins first (Fig. 6e), and later also contain starch grains (Fig. 6g,h). At this late stage, lipids are still present within the endosperm cells (Fig. 7d).

Seed germination is epigeal and emergence of the bright orange radicle, typically 8–12 months after sowing (20–24 months after fertilization), is dramatic (Fig. 5). Approximately 1 month after the radicle penetrates the seed coat, the two cotyledons (along with the hypocotyl and epicotyl), which until this point remained inside the seed, emerge and become fully photosynthetic (Fig. 8a,b). External changes to the seed during the year following dispersal involve rotting of the exotesta and release of a yellow lipid-like layer (Fig. 5a,c,e,g).

Comparative aspects of the embryology of Austrobaileya scandens

The basic embryological features of A. scandens largely correspond with those of other members of the Austrobaileyales. At anthesis, the monosporic female gametophyte of A. scandens is four-celled and four-nucleate, as first reported by Tobe et al. (2007), and similar to what has been found in species of Trimeniaceae (Bachelier & Friedman, 2011), Schisandraceae (Yoshida, 1962; Friedman et al., 2003), and Illiciaceae (Solntseva, 1981; Williams & Friedman, 2004). After fertilization, the diploid endosperm of A. scandens is ab initio cellular and exhibits a marked bipolar pattern of development (sensu Floyd & Friedman, 2000), as in other members of the Austrobaileyales (Hayashi, 1963a,b; Kapil & Jalan, 1964; Floyd & Friedman, 2001; Yamada et al., 2003). Interestingly, we discovered an ephemeral stage during seed development when maternal starch reserves were present in the nucellus, somewhat akin to what might be expected in the very earliest stages of the development of a perisperm – as would be the case in the Nymphaeales, for example. However, these carbohydrate deposits in A. scandens are remobilized out of the nucellus, which is eventually crushed by the rapidly expanding endosperm, where embryo-nourishing reserves will be sequestered through the time of seed maturation and post-dispersal development.

Ephemeral starch reserves in the nucellus of developing seeds have previously been referred to as a transitory perisperm (Juel, 1907). However, this phenomenon has only been documented in a handful of flowering plant taxa. Among early divergent lineages of angiosperms, a brief deposition of carbohydrate reserves in the nucellus has been reported in the seeds of Sarcandra (Chloranthales; Endress & Igersheim, 1997), as well as the ovules of Trimenia (Austrobaileyales; Friedman & Bachelier, 2013), but not in Schisandraceae, Illiciaceae or Amborellaceae (Floyd & Friedman, 2000, 2001). A careful re-examination of histological sections from previous embryological work on Amborella revealed small numbers of small starch grains deposited in the chalazal nucellus of ovules (Fig. S2). These starch grains disappeared shortly after fertilization. By contrast, a persistent starch-laden nucellus (perisperm) is present in all members of the Nymphaeales (Friedman et al., 2012; Povilus et al., 2015).

If perisperm is apomorphic among early angiosperm lineages (see Doyle et al., 1994; Friedman, 2008 and Friedman et al., 2012 for discussion of alternatives), the transient presence of modest starch reserves in the nucellus of various members of ancient clades of angiosperms suggests that, at the very least, a fleeting accumulation of starch in the nucellus may be plesiomorphic. Such modest deposits of starch in the nucellus, as recorded among members of early divergent lineages of angiosperms, may thus have served as a developmental springboard for the evolution of a fully fledged perisperm in the common ancestor of Nymphaeales. Alternatively, should perisperm prove to be the plesiomorphic condition for storage of embryo-nourishing reserves in angiosperms, the transient presence of starch in the nucellus of Amborella, Sarcandra, Trimenia, and Austrobaileya might reasonably be interpreted as a developmental remnant of the transfer of storage of embryo-nourishing reserves to a fully fledged endosperm.

Cells of the mature endosperm in A. scandens accumulate significant quantities of starch (as previously reported by Endress, 1980), as well as some lipids and proteins. By contrast, proteins and lipids constitute the main storage compounds within seeds of Trimeniaceae (Kapil & Jalan, 1964; Prakash, 1998; Friedman & Bachelier, 2013), Schisandraceae (Kapil & Jalan, 1964), and Illiciaceae (Floyd & Friedman, 2000, 2001). Thus, the starch-bearing endosperm of A. scandens is unique among Austrobaileyales. Starch is scarce in or absent from the endosperm of Amborella (Floyd & Friedman, 2001) and members of the Nymphaeales (Cook, 1902, 1906, 1909; Seaton, 1908; Khanna, 1964a,b, 1965, 1967; Floyd & Friedman, 2000, 2001; Friedman, 2008; Rudall et al., 2008, 2009; Friedman et al., 2012; Povilus et al., 2015). These data point to the accumulation of huge quantities of starch in the endosperm as an apomorphic feature of Austrobaileya.

Ontogenetic contrasts among seeds of the most ancient lineages of angiosperms

All members of the Austrobaileyales disperse albuminous seeds with a minute dicotyledonous embryo and a well-developed endosperm that occupies the majority of the seed volume. What is notable about Austrobaileya, among members of the Austrobaileyales, is the extremely long time that elapses between fertilization and seed dispersal, and between seed dispersal and seedling establishment, as well as the large size of the seeds themselves (Fig. 9). By definition, after being shed from the maternal sporophyte, the embryos of all albuminous seeds must undergo further development before germination. This is a direct consequence of ‘incomplete’ embryo development before seed dispersal (Grushvitzky, 1967), which is often synonymously (and perhaps confusingly) referred to as ‘seed maturation’. While germination delays associated with embryo maturation among taxa with albuminous seeds have been referred to as a form of ‘morphological dormancy’ (sensu Baskin & Baskin, 2004), it is worth noting that there is nothing necessarily ‘dormant’ about these seeds. In Austrobaileya, during the period of time between seed dispersal and visible manifestations of seedling germination, the embryo will continue to differentiate, grow, and eventually fill the endosperm chamber.

As members of the earliest diverging clades of angiosperms produce albuminous seeds (Forbis et al., 2002; Finch-Savage & Leubner-Metzger, 2006; Feild, 2008; Linkies et al., 2010; Willis et al., 2014), it is not surprising that the time between seed dispersal and seedling germination is typically not brief in these taxa. It can range from c. 2 to 3 months in Schisandra (Grushvitzky, 1967; Nikolaeva et al., 1985; Zhou & Wang, 2000; Chien et al., 2011) to 3 months in Amborella (Fourcade et al., 2015; Fogliani et al., 2017), and 4 months in Illicium (Thien et al., 1983; Olsen & Rutter, 2001). Seeds of Nymphaeales show a much more variable time to germination. In tropical species such as Nymphaea thermarum, germination occurs in a matter of days (Fischer & Magdalena-Rodriguez, 2010; Povilus et al., 2015). Temperate species of Nymphaeales such as Nuphar lutea and Nymphaea alba go through a period of true dormancy and then germinate in weeks (Beal & Southall, 1977; Smits et al., 1990). In various species of Trithuria, germination occurs within a matter of weeks after seeds are sown (Tuckett et al., 2010; Friedman et al., 2012). Compared with other taxa, the nearly year-long period required for germination in A. scandens is extreme by most measures (Schoonderwoerd & Friedman, 2016). The uniquely large and slow-to-germinate seeds of Austrobaileya are apomorphic within Austrobaileyales (Table 1).

Ecological and evolutionary correlates of large seed size in Austrobaileya

Austrobaileya scandens is a climbing twining liana that grows in remarkably dark (and damp) understory environments of tropical Australia (Bailey & Swamy, 1949; Feild et al., 2003, 2004). Diffuse light intensities measured in undisturbed field environments associated with Austrobaileya seedlings (Feild et al., 2003) are extremely low, ranging from 0.2% to 0.75% of full-intensity sunlight. Thus, seedlings of A. scandens must germinate, grow scandently, and eventually find the support of a tree to climb into the canopy towards higher light intensities (Feild et al., 2003), all while potentially limited by carbon reserves and assimilation. Indeed, Feild et al. (2003) found that, even during the daytime, seedlings of A. scandens may have periods of negative carbon assimilation. All of these factors suggest that large seed size (with correspondingly large nutrient reserves) might be critical to seedling establishment where full and consistent autotrophy by the seedling might not be possible for an extended period of time.

As Westoby et al. (1996, 2002) and others (Moles et al., 2005a,b) have noted, seedlings from large-seeded species are better able to withstand environments with very low light conditions. It is also worth noting that large seed size in Austrobaileya may be linked to its animal dispersal syndrome (cassowaries, which are very large birds, musky rat kangaroos and giant white-tailed rats), a general correlation also reported for tropical plants in Australia (Endress, 1983; Finkelstein & Grubb, 2002; Dennis, 2003; Cooper & Cooper, 2004), and in general for angiosperm seeds (Moles et al., 2005a,b; Eriksson, 2008). Thus, challenges associated with deep understory light conditions and animal dispersal syndromes may both have contributed (in an evolutionary sense) to the extremely large size of Austrobaileya seeds.

Embryo to seed ratios and allometric considerations

Large seed size in A. scandens is pronounced and apomorphic. Interestingly, while the length of the embryo in Austrobaileya is roughly three times that of the embryos of other members of the Austrobaileyales (Table 1) and nearly 50 times that of the embryo in Trithuria submersa (Nymphaeales) (Table 1), the size of the embryo at the time of seed dispersal, as a proportion of overall seed size (embryo:seed length (E : S) ratio sensu Forbis et al., 2002), is not substantially greater (0.08) than that of various species in the Austrobaileyales and other early divergent lineages of flowering plants (Table 1; Fig. 10). In fact, the E : S ratio in A. scandens is almost identical to that of Trithuria submersa (0.09) despite the huge differences in both seed size and embryo size.

Forbis et al. (2002) recovered a hypothetical E : S ratio of 0.16 for the common ancestor of angiosperms, 0.097 for the common ancestor of Nymphaeaceae, and 0.05 for the common ancestor of Austrobaileyales. E : S ratios in Austrobaileyales range from 0.08 (Illicium mexicanum and A. scandens) to 0.231 (Trimenia moorei). In recently described extremely minute (c. 1–2 mm in length) early angiosperm fossil seeds with preserved embryos (Friis et al., 2015a), E : S ratios ranged from 0.11 to 0.18, somewhat higher than but basically similar to what we found in Austrobaileya. Thus, the minute embryo of Austrobaileya and the E : S ratio of 0.08 may signal a developmental and allometric conservatism among clades of early divergent angiosperms and increased seed size may have evolved to allow for greater storage reserves associated with seedling establishment (and possibly coevolved for dispersal by larger animals), but with the maintenance of proportionately small embryo size. Nevertheless, germination time (from seed dispersal to radicle emergence) in Austrobaileya has become decidedly long, probably reflecting the time required for the embryo to grow and fill the endosperm chamber – taking as much as 1 yr in the continuously warm and wet conditions of tropical Australia.

Concluding thoughts

While extant early divergent angiosperm lineages (Amborella, Nymphaeales, and Austrobaileyales) comprise less than one-tenth of a per cent of living flowering plant species, evolutionary diversification within these clades has been significant. Everything from shrubs to small trees, lianas and aquatics can be found among the > 200 species that currently represent these three lineages. From a reproductive perspective, while four-nucleate and four-celled female gametophytes and diploid endosperms predominate (and are plesiomorphic for angiosperms as a whole) (Williams & Friedman, 2002, 2004; Friedman et al., 2003; Friedman & Williams, 2004), a nine-nucleate female gametophyte that yields a triploid endosperm appears to be apomorphic in Amborella (Friedman, 2006; Friedman & Ryerson, 2009). Endosperms are both ab initio cellular (plesiomorphic for angiosperms) and helobial (Cabombaceae) (Floyd & Friedman, 2001). Embryo-nourishing reserves are predominantly lipids and proteins (Amborella, and all members of the Austrobaileyales except Austrobaileya) or starch (Nymphaeales and Austrobaileya) (Floyd & Friedman, 2000, 2001; Friedman, 2008; Friedman et al., 2012; Friedman & Bachelier, 2013; Povilus et al., 2015). Finally, unlike other early divergent lineages of flowering plants that rely on either a diploid or triploid genetically biparental endosperm to nourish the embryo, in the Nymphaeales, perisperm, a maternal sporophytic tissue, serves as the primary embryo-nourishing tissue. The endosperm among water lilies is minute and unassociated with storage of embryo-nourishing reserves (Friedman, 2008; Friedman et al., 2012).

As we have shown in this study, seed size has also undergone tremendous diversification among Amborella, Nymphaeales, and Austrobaileyales (see also Feild, 2008). While it is evident that the earliest flowering plants produced extremely minute seeds (Friis et al., 2015a) with E : S ratios between 0.1 and 0.2, Austrobaileya has, at some point in its evolutionary history, undergone a radical increase in seed size, while retaining the low E : S ratio characteristic of early divergent lineages of angiosperms. The E : S ratios of members of extant early divergent angiosperms, as well as ancient flowering plant fossil seeds, thus appear to be relatively conservative or constrained. Thus, at the time of fruit/seed dispersal in A. scandens, the relatively minute embryo must undergo significant growth and development before it can fill and then escape the confines of the seed.