A study of ovule-to-seed development in Ceratiosicyos ( Achariaceae ) and the systematic position of the genus

A light microscope study of developing ovules and seeds of Ceratiosicyos laevis (Thunb.) A.Meeuse was undertaken to aug­ ment an investigation of ovule and seed structure in Achariaceae. a tri-generic family comprising three species of herbaceous perennials endemic to southern Africa. Tests for myrmecochory suggest that seed of Ceratiosicyos Nees is not dispersed by ants like those of Acharia Thunb. and Guthriea Bolus. Structural differences include the absence of a raphal ridge and imbibition lid and the presence of long funicles and medium-sized embryos ii


INTRODUCTION
Ceratiosicyos laevis (Thunb.)A.Meeuse is one of three species of herbaceous, dicotyledonous perennials that make up an entire family of southern African en demics, the Achariaceae.The family is regarded as high ly modified and its relationships have been much debat ed (see e.g.Bernhard 1999; Steyn et al. 2001 and refer ences therein).Traditionally, Achariaceae were placed among families belonging to Violales (Dahlgren 1980).Based on evidence from phylogenetic analyses of mole cular data (Savolainen et al. 2000) the family is placed in Malpighiales, where it has been linked with Kiggelariaceae which consists of woody perennials from southern and East tropical Africa (Kiggelaria L.), Assam and Burma (Gynocardia R.Br.) and Sri Lanka and Malaysia (Trichadenia Thwaites).
In niche preferences and vegetative morphology, the three herbaceous species of Achariaceae are so diverse (Dahlgren & Van Wyk 1988) that they were placed in separate genera.Yet. in breeding habit and floral struc ture Ceratiosicyos laevis shares many characters with Acharia tragodes Thunb.and Guthriea capensis Bolus.Notable similarities include the presence of few-flowered inflorescences containing both male and female flowers, absence of rudimentary organs of the opposite sex in the unisexual flowers, sympetaly (petals loosely coherent in Ceratiosicyos Nees), conspicuous (yellow ish), antipetalous floral glands, and anthers with broad connectives and unusual, swollen trichomes (Dahlgren & Van Wyk 1988;Bernhard 1999).In addition, recent reproductive biological studies in Achariaceae have shown that ovules and seed characters in Acharia Thunb.and Guthriea Bolus are remarkably similar (Steyn et al. in press).Furthermore, both genera are myrmecochorous and their seeds have the same unusual adaptations for seed germination and dispersal, namely an imbibition lid and a pronounced raphal ridge to serve as a handle for carrying the smooth seed.
For Ceratiosicyos, very little information is available on ovule and seed structure or seed dispersal.A brief report on ovule structure by Bernhard (1999) and a refer ence to seed coat structure (exotegmy, according to Dahlgren & Van Wyk 1988) suggest important embryological differences between Ceratiosicyos and Acharia or Guthriea.It is not known whether Ceratiosicyos also forms part of the herbaceous myrmecochorous flora of southern Africa-it does not bear its fruit near the ground like Acharia and Guthriea for easy collection by ants, but is a vigorously growing, nontendrilliferous twiner that reaches considerable heights along streams at the edge of Afromontane forest, particularly along the east ern escarpment of southern Africa.
For the present study we investigated embryo sac for mation.mature ovule characters, ovule-to-seed develop ment and mature seed and seed coat structure in Ceratiosicyos.We also tested the seed for possible dis persal by ants.Results are compared with those recently obtained on Acharia and Guthriea (Steyn et al. 2001;Steyn et al. in press) to determine the embryological char acters of the family and to evaluate our findings in the light of available embryological data on Kiggelaria L.

MATERIAL AND METHODS
Floral buds, mature female flowers and developing fruit of Ceratiosicyos were collected in Eastern Cape from a population grow ing on the banks of the Maitland River, in the Maitland River Forest Reserve (voucher specimen: Van Wyk 13555 PRU).Additional material that included seeds at dispersal stage was gathered at Kowyns Pass near Graskop in Mpumalanga (voucher specimen: Steyn 24 PRE).All flowering and fruiting stages were immediately immersed and stored in a 0.1 M cacodylate-buffered solution (pH 7.4) containing 4% formaldehyde and 2.5°7c gluteraldehyde.Flowers and fruits were later dissected, ovules and developing seeds removed, sorted according to size and rinsed in the buffer.Dehydration and impregnation with glycol methacrvlate (GMA) followed the methods of Feder & O 'Brien (1968).A selection of impregnated structures was individually imbedded in GMA, hardened in the oven at about 58°C and sectioned transversely and sagittally.Selected sections were stained with the periodic acid/Schiff reaction and counterstained with toluidine blue O by using the protocols of O' Brien & McCully (1981).
Tests for myrmecochory: at the collection site on the Kowyns Pass, dehiscing capsules were carefully removed from the plants, the seeds collected and immediately strewn onto the trails or near the nests of ants of varying sizes found at the collection site.Mature capsules were stored in air-tight bags overnight and taken to Pretoria where seeds, removed from capsules that had in the mean time split open, were again offered to ants.Dried-out seeds with tuberculate surfaces were also offered to ants.

Placentation and orientation o f ovules
The hypogynous female flowers of Ceratiosicyos contain elongated, pentagonal ovaries borne on gynophores.The ovaries are unilocular and usually contain 7-15 anatropous ovules that are borne singly on five parietal placentae.The latter regions are not ridged and run longitudinally along the inner surface of the ovary wall, opposite the five median carpel traces.Ovules from five placentae are so arranged that they form a single row in the narrow locule and this alignment is maintained throughout ovule-to-seed development.The alignment is achieved by the ability to vary the orientation of the ovules and the lengths of the funicles-some ovules point upwards, others downwards and the funicles may be short or more than twice the length of the ovular body to place the ovules neatly in a single row.

Structure o f mature ovule
Mature ovules are anatropous, bitegmic, crassinucellate structures (Figure 1A) with an ovoid shape and ± 520 |jm long (funicle excluded).The integuments are multilayered, the outer consists of four to five layers in its central part, while the inner is up to seven layers thick in this region.The outer epidermis of the inner integu ment is very conspicuous-the cells are four to six times longer than any other cell in the ovule w ith the exception of the embryo sac.On the antiraphal side of the ovule, the tip of the outer integument increases in thickness by tangential divisions in its inner epidermal layer.In pre fertilization stages of ovules, the outer integument is as long as the inner integument so that the micropyle canal is formed by the inner integument only.The raphe is not ridged.The vascular bundle of the raphe branches as soon as it enters the chalaza.but the integuments are not vascularized (Figure 1A).
The mature embryo sac is about one-third the length of the nucellar cylinder and lies in the centre of massive nucellar tissue.About six layers of nucellus cells cover the embryo sac on all sides.Below the micropyle.at least three of these layers result from periclinal divisions of the nucellus epidermis (Figure 1C.D).The nucellus apex is slightly attenuate, but does not protrude into the micropyle.
The embryo sac develops from the chalazal dyad cell while the micropylar dyad cell degenerates (Figure IB).During the second meiotic division the chalazal dyad is not partitioned by a transverse cell wall so that both megaspore nuclei are included in the same cell (Figure IB).After two mitotic divisions an eight-nucleate, bisporic embryo sac of the Allium Type is formed.The mature embryo sac contains many starch grains.A small egg apparatus occurs below the parietal nucellar tissue (Figure 1C).The short neck regions of the synergids con tain a filiform apparatus.A large central cell nucleus lies in about the central part of the embryo sac, while three ephemeral antipodal cells (not shown) develop in the elongated and narrow chalazal base (Figure 1A) of the embryo sac.

Early development o f endosperm and embryo
Fertilization is porogamous in Ceratiosicyos and endosperm formation is nuclear.After entering the micropyle.the tip of the pollen tube swells and stains darkly with PAS and toluidine blue (Figure 2A).During the initial stages of embryo sac enlargement (Figure 2A), free endosperm nuclei become arranged in a single layer alongside the embryo sac wall.When the growing seed has reached a size of ± 5 x 2.5 mm. the first cell walls are laid down between adjacent endosperm nuclei, and the embryo sac then gradually becomes filled, layer upon layer, with thin-walled endosperm cells.
The zygote remains inactive during the nuclear stage of endosperm formation.The first division of the zygote was not seen, but pro-embryos in the tetrad stage of develop ment (Figure 2B) were found before the endosperm start ed to become cellular.These four-celled pro-embryos are T-shaped which shows that the apical cell (ca) has divid ed in a vertical plane and the basal cell (cb) transversely (Figure 2B).The two daughter cells of ca then both divide obliquely (Figure 2C) so that a bicellular, wedgeshaped epiphysis (e) is formed in the apical tier during the quadrant stage of the pro-embryo (i.e.w hen the deriv atives of ca comprise four cells).The epiphysis later forms the shoot apex, w hereas the remaining cells of the quadrant form the coty ledons (Natesh & Rau 1984: 390).The pro-embryo is globular in shape and has no suspensor.Below the cells of the quadrant, the uppermost deriva tive of cb is a discoidal cell (h).designated 'hypophysis' by Hanstein (1870).The hypophysis later forms the ini tials of the root cortex and the root cap (Crété 1963).
In Johansen's (1950) classification of e m b r y o g e n ic types.T-shaped pro-embryos are characteristic of both the Onagrad Type and Asterad Type, but it is only in the latter type that the basal cell (cb) contributes significant ly to the formation of the embry o proper.Ceratiosicyos embryos have no suspensors.all derivatives of the basal cell are incorporated into the embryo proper which indi cates an Asterad Type embryo.The presence of the epi physis also points tow ards the Asterad Type-it is in taxa conforming to this type that an epiphysis is formed dur ing the quadrant stage (Natesh & Rau 1984: 386. fig. 8.6).The lack of a suspensor places the embryo of Ceratiosicyos in the Penaea variation of the Asterad Type (Natesh & Rau 1984: 390. 414).

Development and structure o f the seed coat
The seed coat of Ceratiosicyos is mainly derived from the outer integument.This integument forms the sarcotestal layers and the outer, wavy layer of sclereids that protrude peak-like into the sarcotesta (Figures 3D: 5D).The inner integument contributes a few layers of periclinally elongated fibres to the seed coat.During seed coat development the cuticle between the outer and inner integument gradually disappears so that a study of the mature seed coat alone does not show which part each integument plays in the formation of the seed coat.

Contribution of the outer integument (testa) to the seed coat
In pre-fertilization stages, the outer integument con sists of about five cell layers (Figure 1A), except at its rim where the number of layers increases through tangen tial divisions of the inner epidermis.The cells in the rim remain meristematic and in early post-fertilization stages the distal part of the outer integument grows beyond the inner integument to take part in the formation of the micropyle (Figure 2A).In developing seeds a layer of actively dividing endotestal cells (s) can be seen inside the developing sarcotesta (Figure 3A, B, D).These cells are the derivatives of the inner epidermis of the outer integument.At first, the derivatives lie in radial rows and the layer is of even thickness (Figure 3A, B).When the seed has reached its final length of ± 6 mm, the endotestal layer becomes wavy and starts forming pro jections into the sarcotesta (Figure 3D).The endotestal cells later develop into closely packed, thick-walled sclereids with starch grains and single, large crystals of cal cium oxalate in some of the cells, but the crystal-containing cells do not form a continuous layer.At seed dis persal stage, the contents of the endotestal sclereids stain intensely with toluidine blue, indicating the presence of phenolic substances (Figures 3C; 4A, B).
The epidermis and mesophyll of the outer integument form a sarcotesta that envelops the whole seed, including the chalaza and raphe.The raphal region is not pro nounced, i.e. a raphal ridge is not formed (Figures 4A;  5D).The raphal bundle lies imbedded in thin-walled.parenchymatous, sarcotestal tissue that, especially in this area, contains large numbers of starch grains (Figure 4A).Stomata, not seen in the outer epidermis of the ovule, were found at regular intervals in the developing (Figure 3A, B) and mature (Figure 3C) epidermis of the sarco testa.
When the seeds are dispersed, the cells of the inner most layer of the sarcotesta have developed small, fibril lar protuberances on their inner tangential walls (Figures 3C; 4A, B).The fringe-like wall ingrowths are strongly PAS-positive and also stain dark blue with toluidine blue.The fringe layer possibly represents a layer of transfer

Contribution of the inner integument (tegmen) to the seed coat
After fertilization the conspicuously elongated outer epidermal cells of the inner integument (Figure 1 A) ini tially divide anticlinally to form a single layer of dense ly packed, radially elongated meristematic cells (Figure 3A).While the first divisions of the pro-embryo are tak ing place, these meristematic cells of the tegmen divide periclinally once or twice to form three to four layers of cells that are stretched in a direction parallel to the lon gitudinal axis of the seed (Figure 3B).At this stage the cuticle between the developing endotesta and exotegmen starts to disappear, but in longitudinal sections of imma ture seeds the boundary between the two layers is clear because of the difference in the orientation of the cells (Figure 3B, D).When the seed reaches its final size, the exotegmic cells contain many starch grains and the outer elements have started maturing into fibres (Figure 3D).At seed dispersal stage the fibres have thick, lignified walls with simple pits and contain no starch (Figures 3D: 4A, B).
The mesophyll and inner epidermis of the tegmen do not play a significant role in the structure of the mature seed coat.The thin-walled mesophyll tissue initially shows divisions in various planes so that the layers increase in number (Figure 3B).The innermost layer of mesophyll cells becomes conspicuous by their large size and darkly staining properties (Figure 3B.D).At first, the inner epidermis of the tegmen keeps pace with the growth of the seed by dividing anticlinally so that a layer of small, densely packed cells is formed (Figure 3B).Eventually, when the seeds are dispersed, all layers inside the exotegmic fibres are obliterated and a struc tureless pellicle remains between the fibres and the flat tened cells of the nucellar tissue (Figures 3C; 4A).

Macromorphology o f the fruit and seed and tests for myrmecochory
Mature fruit consists of 50-90 mm long.5-valved cap sules (Figure 5A, C) that are thin-walled and light green at seed dispersal stage.Five to twelve stalked seeds are arranged in a single row, sometimes packed close to one another in the locule.When the valves split open (not very forcefully), the seeds break off, leaving their funicles of various lengths attached to the centre of the valves (Figure 5C).Seeds are ovoid to short-cylindrical, 6.0-6.5 mm long (Figure 5B), dark green to brown and covered with a thin, succulent and translucent sarcotesta.The seed surface becomes tuberculate when the sarcotesta dries out, reflecting the projections formed by the underlying mechanical layers (Figure 5D).At seed dispersal, the axile embryo (sensu Martin 1946: 520) is of medium size (i.e. it occupies about three-quarters of the length of the endosperm).It lies straight in the seed, has thin, spatulate cotyledons and a well-formed radicle (Figure 5D).Tests for myrmecochory were negative.If ant-seed interactions are not species specific as claimed by Slingsby & Bond (1981) and found by Steyn et al. (in press), our results suggest that Ceratiosicyos seeds are not dispersed by ants.

Differences betw een Ceratiosicyos and Acharia and Guthriea
A comparison of ovule and seed characters in Ceratiosicyos w ith those of Guthriea and Acharia (Table 1) shows that the three genera are fundamentally very similar in characters usually regarded as of taxonomic importance (see No. 1,2,[8][9][10][11][12]14. 16 & 17).The struc ture of the integuments is also comparable to a large degree, although Ceratiosicyos lacks the peculiar zigzag micropyle (see No. 5) that characterizes ovules and seeds of Guthriea and Acharia.The short outer integument in the Ceratiosicyos ovule possibly does not denote an important structural difference with the other two genera, because this integument overtops the inner after fertiliza-  tion.The inner integument of Ceratiosicyos bulges into the micropyle canal (Figure 2A).The bulging cells might have been mistaken for a nucellar beak by Dahlgren & Van Wyk (1988).The present investigation also showed that the mechanical seed coat layer in Ceratiosicyos is, like those of the other two genera, of dual origin (endotestal-exotegmic) and not purely exotegmic as pre viously reported (Dahlgren & Van Wyk 1988).
Many of the differences between Ceratiosicyos and the other two genera can possibly be attributed to specif ic adaptations for seed dispersal and germination (see No. 6,7,13,15,18 & 19).Ceratiosicyos is not myrmechorous, the ovule therefore lacks the pronounced raphe that, in Acharia and Guthriea, eventually forms a ridge like part of the sarcotestal elaiosome (see No. 6 & 19).The presence of unicellular hairs on the seed surface of Guthriea and Acharia (see No. 15) possibly also relates to myrmechory, since openings left by broken-off trichome bases would allow ant-attracting substances to rapidly reach the seed surface (Steyn et al. in press).
Instead of a pronounced raphe, ovules and seeds of Ceratiosicyos might have developed long funicles as an adaptation to seed dispersal (see No. 7).By varying the lengths of the funicles, the seeds can be manipulated into a single, vertical row in the narrow, elongated locule.This arrangement may be necessary for rapid splitting of the fruit by distributing pressure on the valves evenly along the length of the locule.In the two diplochorous (autochorous + myrmecochorous) genera (see No. 19) the seeds are sessile in the short-cylindrical capsules and pressure on the valves is applied by the swollen elaiosomes.Also, the capsules remain within the covering of persistent corolla tubes to protect developing seeds with their soft elaiosomes (Steyn et al. in press).Ceratiosi cyos is autochorous; its seeds need less protection and developing capsules rapidly outgrow the protective cov ering of the corolla tubes.
Seed size in genera of the Achariaceae does not differ significantly, but Ceratiosicyos has a much larger embryo than the other two genera (see No. 11 & 13).We propose that the small embryo has been the causal factor for the formation of the unique seed lid in the other two genera.This device would allow water and air to enter the seed through the unsclerified cells in the rim of the lid during the slow maturation (12 weeks) of the embryo in the hydrated seed (Steyn et al. in press).The much larger embryo of Ceratiosicyos possibly did not require such an adaptation.Martin (1946) reasoned that smallness in embryos, as compared to size of the endosperm, is representative of a primitive state in seeds of angiosperms, and, conversely, that embryos which become well developed before dor mancy, reflect a higher evolutionary rank.For dicotyle dons, Comer (1976: 48) also regarded small embryos as primitive and considered simplification through loss of structures or cell layers as an indication of an advanced state in seeds.However, considered on its own, it is usu ally difficult, if not impossible, to tell which of the two embryo states-small or large-is the more primitive for a particular taxon.One reason for this difficulty is that embryo (and seed) size, like so many other characters.are subject to homoplasy (parallelism, convergence and character state reversals).Unfortunately homoplasy is rampant among seed plants, thus considerably limiting the reliability of outgroup comparisons to establish polarity (Cronquist 1988).Compared to the other mem bers of the family, Ceratiosicyos shows a notable trend towards simplification of the seed through the loss of several cell layers and structures, e.g.trichomes, a bi-layered testa epidermis, a hypodermis, a crystal-containing layer inside the fringe layer, perisperm and the reduction of the chalazal region with seed lid.These reductions, together with the larger embryo, may indicate that Ceratiosicyos is more advanced than Guthriea and Acharia.On the other hand, indications are that Kiggelariaceae may well be the sister group of Achariaceae (see further on).Both Kiggelaria africana and Ceratiosi cyos laevis share a medium-sized embryo and rather unspecialized seed, coat, states that may be the plesiomorphic ones in Achariaceae.Relatively small em bryos and a rather elaborate seed coat occur in Acharia tragodes and Guthria capensis, both having specialized myrmecochorous seeds.Therefore Ceratiosicyos laevis, with its lianeous habit and mesic forest habitat, could just as well be the more primitive member of the family.Acharia tragodes (semi-woody shrublet) is confined to the xerophytic thicket vegetation of the Eastern Cape, with Guthria capensis (rosulate herb) confined to tem perate grassland and karroid vegetation at high altitude.

Achariaceae versus families in Malpighiales
A detailed comparison of ovule and seed characters in Achariaceae and the 36 families placed in Malpighiales (including many families traditionally placed in Violales) by Savolainen et al. (2000), is hampered by a lack of comparable data for many of the families (Davis 1966;Johri et al. 1992;Nandi et al. 1998).Achariaceae seem generally well placed in Malpighiales and fit in comfort ably among those families previously regarded as part of Violales sensu Dahlgren (1980).Similarities include bitegmic, anatropous, crassinucellate ovules, parietal nucellus partly formed by nucellus epidermis deriva tives, both integuments participating in formation of micropyle canal, nuclear endosperm becoming copious in the seed and a medium-sized to large embryo lying straight in the seed.Some of the characters observed in Achariaceae are rare for Malpighiales, namely a bisporic Allium Type embryo sac, suspensorless Asterad Type embryos, pro tective seed layers containing endotestal sclereids and exotegmic fibres, and a sarcotesta.A number of charac ters shared by Guthriea and Acharia, such as the zigzag micropyle, distally lobed outer integument, perisperm and small embryo is also uncommon for the order.A zigzag micropyle and Asterad embryos conforming to the Penaea variation only occur in Violaceae (Davis 1966), while lobed integuments and perisperm are found in Scyphostegiaceae (Van Heel 1976;Johri et al. 1992) and sarcotestal seeds in Passiflorales (Nandi et al. 1998).
A possible phylogenetic link between Achariaceae and Flacourtiaceae (tribe Pangieae, particularly Kiggelaria africana) was first suggested by the breeding behaviour of a butterfly.Several butterflies in the sub tribe Acraeina (subfamily Acraeinae, tribe Acraeini), including the common garden acraea (Acraea horta), uti lize as larval food plants, members of a closely knit group of plant families traditionally classified in the order Violales (notably Achariaceae.Flacourtiaceae, Passifloraceae and Turneraceae), all containing a unique group of toxic compounds known as cyclopentenoid cyanogenic glucosides (Seigler 1975;Dahlgren 1980;Cronquist 1981;Takhtajan 1997;Kroon 1999).In their natural habitat larvae of Acraea horta feed mainly on Kiggelaria africana.a species containing gynocardin as major cyanogenic glucoside (Jaroszewski & Olafsdottir 1987;Raubenheimer & Elsworth 1988).The larvae selectively sequester and store some of the gynocardin, which are passed on to all other stages in the life cycle, supplemented by apparent self-synthesis (Raubenheimer 1987(Raubenheimer . 1989).Accumulation of this toxin is believed to render the insects unpalatable to predators.Previously gynocardin has been isolated from the seed of Gynocardia odorata R.Br., another member of the tribe Pangieae (Coburn & Long 1966).When live plants of Ceratiosicyos laevis and Guthriea capensis were intro duced into the botanical garden at the University of Pretoria in the mid-1980s, both were immediately select ed by Acraea horta for egg deposition; larvae emerged and butterflies were raised (Dahlgren & Van Wyk 1988).This observation led to a chemical study of Ceratiosicyos laevis, the first of its kind a member of the Achariaceae, resulting in the identification of gynocardin as one of the principal cyanogenic glucosides in this species (Jensen & Nielsen 1986); its presence in Guthriea capensis is suspected.
Based on evidence from molecular biology, Chase et al. (1996) also suggested a linkage between the herba ceous Achariaceae and the woodv tribe Pangieae.This tribe included amongst others.Gynocardia R.Br.. Hvdnocarpus Gaertn.. Kiggelaria L.. Pangium Reinw.and Trichadenia Thwaites (Lemke 1988).In the circumscrip tion of Soltis et al. (2000), Kiggelariaceae include Pangium.Hydnocarpus, and Kiggelaria.Our results show that Achariaceae agree closely with the latter two genera as far as seed development and seed coat structure are concerned.Van Heel (1979) found the seeds of Hydnocarpus and Kiggelaria sarcotestal with undulating endotestal-exotegmic mechanical layers, a dominant endotestal layer of sclereids.an outer integument that is initially short, but overtops the inner during seed forma tion and a cuticle that disappears early so that the dual nature of the protective layer is masked and the erro neous impression given that the seeds are pachychalazal.These characters are so similar to the characters we found in Achariaceae that they can be regarded as strong support for a linkage between Achariaceae and Kigge lariaceae.
In Kiggelaria the embryo is of medium size, as found in Ceratiosicyos.It is perhaps noteworthy that seeds with a small embryo, a fleshy raphe and a conspicuous notched cuticle between the tegmen and nucellus as reported for Guthriea (Steyn et al. 2001) also occur in Berheridopsis Hook.f.(Van Heel 1979).This taxon.pre viously also included in the Flacourtiaceae (Lemke 1988), is currently regarded as a relict with an unclear taxonomic position (Savolainen et al. 2(XX)).

FIGURE 1 .
FIGURE 1.-Ovule and embryo sac of Ceratiosicyos as seen in sagittal section.A, mature ovule; B, early stage in development of bisporic embryo sac; C, D, consecutive sections of same ovule as in A to show details of mature embryo sac.a, egg cell; c, central cell nucleus; curved arrows indicate megaspore nuclei in chalazal dyad cell; d, disintegrating micropylar dyad cell; n, derivatives of nucellus epidermis; r, outer epidermis of inner integument; v, vascular bundles in chalaza.Scale bars: A, 100 pm; B, 10 ym; C, D, 50 pm.

FIGURE 2 .
FIGURE 2.-Early development of endosperm and embryo in Ceratiosicyos.A, developing seed during resting stage of zygote; B, T-shaped pro embryo; C, suspensorless pro-embryo during quadrant stage, ca, apical cell after vertical division; cb, basal cell after transverse division; e, epiphysis; h, hypophysis; i, inner integument; 1, bulge of inner integument; o, outer integument participating in formation of micropyle canal; t, pollen tube in elongating embryo sac.Scale bars: A, 200 jam; B-C, 50 pm.

FIGURE 3 .
FIGURE 3.-Development and structure of seed coat in Ceratiosicyos.A, t/s young seed coat just after fertilization: B, 1/s seed coat during first stages of pro-embryo formation; C, 1/s mature seed coat of dispersed seed: D, 1/s seed coat during maturation of fibrous exotegmen.Curved arrows indicate position of stomata; n, nucellus cell remains; p, inner epidermis of tegmen; r, outer epidermis of tegmen with anticlinal divisions; s, derivatives of inner epidermis of testa; u, sarcotesta: y, mesophyll of tegmen.Scale bars: A, 50 pm: B, C, 100 pm; D, 200 pm.

FIGURE 4 .
FIGURE 4.-Development and structure of seed coat of Ceratiosicyos (continued): A, raphe and underlying seed coat layers in a median t/s of seed: B, t/s fringe layer and adjacent cell layers seen at higher magnification than in Figure 3C.f, fringe layer; r, fibres of exotegmen; s, derivatives of inner epidermis of testa; u, sarcotesta.Scale bars: A, 200 jam; B, 50 pm.cells (Gunning & Pate 1969), often found in reproductive structures for the short-distance transport of solutes (John & Ambegaokar 1984: 29, fig.1.13A-F).