Embryology and systematic relationships of Kiggelaria ( Flacourtiaceae )

Kiggelaria L. is endemic to Africa and the only representative o f tribe Pangieae (Flacourtiaceae) on the continent. Molecular genetics, phytochemistry and host-parasite relationships have indicated a relationship between this woody, pantropical tribe of Flacourtiaceae and a small, trigeneric family of herbaceous, southern African endemics, the Achariaceae. In the present study, ovule and seed structure in Kiggelaria were investigated and compared with relevant data recently reported for Achariaceae. Support for an alliance with Achariaceae were found in the presence of anatropous, bitegmic. sessile ovules with zigzag micropyles. deep-lying embryo sacs covered by an epistase in the ovule and seed, endotestal-exotegmic seed coat layers, suspensorless embryos and sarcotestal seed with a prominent, plate-like hypostase. Unlike Achariaceae. Kiggelaria seeds do not have chalazal seed lids, pronounced raphal ridges, a fringe layer, or stomata in the outer epidermis o f the sarcotesta. Structural dissimilarities in seeds of omithochorous Kiggelaria and myrmecochorous Achariaceae were regarded as adaptations linked to different strategies for seed germination and dispersal.


INTRODUCTION
Kiggelaria L. is a widely distributed, wholly African genus of dioecious trees and shrubs.It occurs in all the provinces o f the Republic of South Africa and in Lesotho, Swaziland.Namibia, south tropical Africa and tropical East Africa as far north as Mount Kilimanjaro (Killick 1976a).In these areas Kiggelaria represents an important floristic element of forest in the archipelago like Afromontane Region of Endemism (White 1978).This African phytocorion is of special biogeographical and evolutionary significance because of its putative ancient status.Although the genus shows considerable variability in juvenile and mature vegetative morpholo gy.only one polymorphic species is currently recog nized.namely Kiggelaria africana L. (Killick 1976b).
In most modem suprageneric classification systems of Flacourtiaceae.Kiggelaria is placed within the tribe Pangieae.This tribe of about 10 genera has a disjunct, pantropical distribution (Wendt 1988) with most genera reported from Malaysia (Chase et al. 2002).Chiangiodendron T.Wendt occurs in southeastern Mexico (Wendt 1988), Baileyoxylon C.T.White is found in Australia and Kiggelaria is the only representative from Africa.Al ternatively.Kiggelaria and other genera of the Pangieae have been treated as a separate family.Kiggelariaceae (Savolainen et al. 2(KX): Soltis et al. 2(XX)).Within Flacourtiaceae.the Pangieae form a more or less homo geneous group that, in terms o f generic content, has remained consistent in taxonomic treatments (Hutchin son 1967;Lemke 1988. Takhtajan 1997).On account of comparative macromorphology, wood anatomy, palynology and the distribution of selected classes of chemical constituents, Lemke (1988)  A possible phylogenetic link between one such fami ly.namely Achariaceae, a family of three monogeneric genera from southern Africa, and the tribe Pangieae.par ticularly Kiggelaria.was first suggested by the breeding behaviour of a butterfly, Acraea horta (Nymphalidae: Heliconiinae: Acraeini) in the botanical garden of the University of Pretoria.This was reported by Dahlgren & Van Wvk (1988: see also Stevn et al. 2002b).Based on evidence from molecular biology.Chase et al. (1996) also suggested a linkage between the herbaceous Achariaceae and the woody tribe Pangieae.More recent phylogenetic molecular data reported by e.g.Soltis et al. (2Ó00) and Savolainen et al. (2000) have provided addi tional support for a close association between Acharia Thunb.. Guthriea Bolus (Achariaceae) and Kiggelaria.The third genus.Ceratiosicyos Nees.was not included in these investigations.Lately.Chase et al. (2002) proposed splitting Flacourtiaceae sensu Lemke ( 1988) and em end ing the circumscription of the families Achariaceae Harms and Salicaceae Mirb.Achariaceae sensu Chase et al. (2002) thus include Acharieae Benth.& Hook.f.. Pangieae (sensu auct.),Erythrospermeae DC. and a newly described tribe.Lindackerieae Zmarzty (in Chase et al. 2002: 172. 173).all cyanogenic.The non-cyanogenic tribes of Flacourtiaceae sensu Lemke [Flacourtieae DC.. Samydeae (Vent.)(Pangieae) as described by Van Heel (1979).This resem blance suggests that a detailed comparison of ovule and seed characters m ight reveal additional sim ilarities between Kiggelaria and Achariaceae.However, embryological data on Kiggelaria obtainable from literature were scant and ambiguous; we therefore re-investigated ovule and seed structure in Kiggelaria africana.The results of our study are given in the present report.

MATERIAL A ND METHODS
Open flowers and developing fruit were collected from female trees in a dioecious, natural population of Kiggelaria africana growing in the National Botanical Garden, Pretoria, South Africa.To facilitate penetration of chemicals into ovules and seeds, the thick ovary wall was partly removed and developing seeds were removed from the locule.All material was fixed and stored in a 0.1 M cacodylate buffered solution containing 4% formalde hyde and 2.5% gluteraldehyde.Following the methods of Feder & O ' Brien (1968), material was dehydrated in an alcohol series and impregnated with glycol methacry late (GMA).All material was imbedded in GMA, sec tioned transversely or longitudinally at 2 -3 jam and sub sequently stained with the periodic acid/Schiff reaction and toluidine blue O (pH 4.4) by using the protocols of O 'Brien & McCully (1981).

Placentation and orientation of ovules
The unilocular, thick-walled and densely pubescent ovaries in female flowers (Figure 1) contain many ovules borne singly or in pairs on four or five parietal placentas.The sessile ovules are supplied by vascular strands con nected to the large vascular bundles lying opposite the petals with their conspicuous, adaxial nectary glands (adnate adaxial basal scales, according to Chase et al . 2002).The position of the placental bundles in relation to the petals suggests that they represent the marginal bun dles of the congenitally fused carpels.During the matu ration of ovules, the space in the locule becomes restrict ed.The ovules are pressed against each other and the ovary wall, and pushed out of alignment with the micropyles pointing in all possible directions.

Structure of mature ovule
Ovules are anatropous, bitegmic, crassinucellate and ovoid in shape (± 530 x 400 pm) when the flowers open.Sagittal sections show that there is no funicle and the raphe is pronounced (Figure 2A).The vascular bundle of the raphe branches as it enters the overgrown chalaza, but the ramifications do not enter the integuments.Both integuments are multi-layered.The outer is thicker than the inner, especially at its distal rim where it overtops the inner integument to take part in the formation of the slightly zigzag micropyle canal (Figure 2A, B).At anthesis, the outer and inner epidermal layers of the outer integument are separated by about four layers of thinwalled, isodiametric mesophyll cells containing many, small starch grains and occasionally showing periclinal as well as anticlinal divisions.Periclinal divisions also occur in the inner epidermal cells of this integument, whereas the cells of the outer epidermis and the epider mis of the chalaza mostly divide anticlinally to form a layer of radially flattened cells (Figure 2A, B).The inner integument is about five layers thick and its cells are smaller than those of the outer integument, except for the outer epidermal cells of the inner integument which are elongate in the direction of the longitudinal axis of the ovule.A distinct cuticle layer separates the integuments from each other.
The ovoid, relatively small nucellus with enclosed embryo sac, lies in the centre of the ovule, occupying  The pear-shaped embryo sac of the mature ovule thus lies deeply imbedded in nucellar tissue.The narrow chalazal end contains three small antipodal cells (Figure 2B).The nucleus of the central cell lies in the centre.adjacent to the wall, whereas the egg apparatus occupies the wider, micropylar region.The position of the two synergids with darkly stained, well-formed filiform apparatus (Figure 2A), clearly indicates the distal limits of the embryo sac.Reports that the tip of the embryo sac of Kiggelaria breaks through the nucellus tissue and enters the micropyle (Johri etal.1992) are not substantiat ed by the present study.

Seed and seed coat development
Fertilized ovules enlarge rapidly and endosperm for mation precedes embryo development.During the freenuclear stage of the endosperm, the zygote remains in a resting phase directly below the micropyle (Figure 2C) and is separated from the endostome by the thick-walled remnants of the nucellus cap that form an epistase.This tissue plugs the micropyle (Bouman 1984) and it persists in later stages of embryo development (Figure 2D).
The embryogeny of Kiggelaria was not studied in detail; pro-embryos were not found in our material and the embryo could not be typified.When the seed reaches its final size of about 6 mm. the free-nuclear endosperm becomes cellular.The embryo then is in the early heart-shaped stage and has no suspensor (Figure 2D).By the time the capsule splits open to release the ripe, bright orange-red seeds, the embryo lies in the centre of the endosperm, has thin, spatulate cotyledons and is of medium size.i.e. it extends about halfway up into the endosperm.

Contribution o f the outer integument (testa) to the seed coat
The outer integument is strongly multiplicative and all cell layers contribute to the formation of the mature seed coat.The outer epidermis divides periclinally once or twice (Figure 3A) and the resulting epidermal tissue eventually acquires unevenly thickened cell walls to form a collenchymatous pellicle (Figure 3B, C).This protective layer also covers the chalaza and raphal region of the seed and contains no stomata.The cells of the mesophyll below the epidermis of the integument, raphe and chalaza divide in various planes to develop into the multi-layered, thin-walled, succulent tissue of the sarcotesta (Figure 3A -C).In the ripe seed, the contents of the innermost cells of the sarcotesta reacts strongly with PAS and toluidine blue.These dark-staining cells form a layer that separates the sarcotesta from the mechanical tissue (Figure 3B The outer integument contributes to the protective, mechanical tissue of the seed coat.This tissue has a dual derivation and consists of two sub-layers.The outer layer originates from the inner epidermis o f the outer integu ment; the inner layer from the outer epidermis of the inner integument (see further on).The cells of the inner epidermis start dividing periclinally just after fertiliza tion to form radial rows o f cells (Figures 2C; 3A).The rows later become disrupted, because the outer cells undergo further divisions in various planes, the inner cells increase in size and the developing exotegmic fibres start intruding into the endotestal layers (Figures 2D; 3B.C).At seed dispersal stage, the large cells of the endotesta have developed into thick-walled, isodiametric sclereids (Figure 3C).The outer, smaller cells of the endotesta remain relatively thin-walled and separate the sclereids from the layer of dark-staining sarcotestal cells (Figure 3C).At the chalazal end of the seed, the layers of rela tively thin-walled, endotestal cells continue into the cha laza.surround the vascular tissue and separate the latter from a thin, plate-like layer of lignified cells that repre sents a hypostase (not illustrated).

Contribution o f the inner integument (tegmen) to the seed coat
The inner sub-layer of the mechanical tissue is exotegmic and originates from the outer epidermis of the inner integument.The cells divide periclinally and cells are formed centripetally (Figures 2C; 3A).The deriva tives rapidly stretch in a direction parallel to the longitu dinal axis of the seed (Figures 2C.D; 3B, C).At this stage the cuticle between the two developing sub-layers is still distinct.When the endosperm becomes cellular, the tips of the elongated exotegmic cells start intruding into the adjacent sub-layer of endotestal tissue (Figures 2D; 3 B ) and the cuticle becomes disrupted.At seed dis persal stage the endotegmic sub-layer has matured into thick-walled, longitudinally orientated fibres (Figure 3C).This sub-layer does not continue into the chalaza.
The mesophyll and inner epidermis of the inner integument do not play a significant role in the structure of the mature seed coat; the layers disintegrate when the endosperm tissue matures (Figure 3C).The cuticle be tween the nucellus and inner epidermis of the inner integument, so prominent in seeds of Guthriea (Steyn et al. 2001). is thin.A pigment layer that, according to Van Heel (1979), differentiates on the inside of the sclereid layer in Kiggelaria (also Hydnocarpus) and persists when the mesophyll disintegrates later on.was not seen during this investigation.

DISCUSSION
For most of the 80-95 genera traditionally placed in Flacourtiaceae (e.g.Hutchinson 1967;Lemke 1988) and recently split in two groups to expand the families Achariaceae and Salicaceae (Chase et al. 2002), embryological characters are completely unknown.The meagre, scattered data used to compile accounts of Flacourtiaceae in compendia dealing with comparative embryology (e.g.Davis 1966;Johri et al. 1992) provide no support for the 'sweeping taxonomic changes' proposed by Chase et al. (2002).However, two embryological studies do suggest that such changes might have merit.Firstly, a recent study on members of Flacourtiaceae sensu lato with a multiwhorled androecium (Bernhard & Endress 1999).showed that the stamens are initiated centrifugally in Flacourtieae and Scolopieae (non-cyanogenic tribes) as reported for Populus in Salicaceae Mirb.(Kaul 1995).In Erythrospermeae.Oncobeae (except Oncoba) and Pangieae (cvanogenic tribes) initiation is centripetal or simultaneous.Achariaceae, however, are characterized by a single whorl of 3-5 stamens (Dahlgren & Van Wyk 1988 1999).Secondly, an earlier work on seed coat structure by Comer (1976) indicated that the family should be divided in two groups, namely a Flacourtia group (Casearia Jacq., Flacourtia L'Hér., Idesia Maxim.. Oncoba Forssk.) and a Hydnocarpus group (Hydnocarpus, Kiggelaria.Pangium Reinw., Scaphocalyx Ridl.).Corner (1976) mistakenly interpreted the seeds of all investigated members of his Hydnocarpus group as pachychalazal with no contribution of the integuments towards the formation of mechanical seed coat layers.Van Heel's (1974, 1979) studies showed that the seed coat of only Pangium is pachychalazal; Erythrospermum Lam.. Hydnocarpus and Kiggelaria have seed coats with endotestal-exotegmic mechanical layers, but the integumental derivation and dual origin of the mechanical lay ers are masked by the early disappearance of the cuticle boundary.In Caloncoba Gilg and Camptostylus Gilg (Lindackerieae) the cuticle separating the inner and outer integument also vanishes entirely during seed formation (Van Heel 1977).Results obtained during the present study confirm Van H eel's (1979)  A second embryological character of Kiggelaria that needed clarification is the shape of the embryo sac.Davis (1966) reported that the embryo sac of Kiggelaria does not become much elongated, while Johri et al. (1992) found the embryo sac in Kiggelaria similar to that of Azara Ruiz & Pav.(= Arechavaletaia Speg.) with an embryo sac breaking through the nucellus and reaching the exostom e of the micropyle.An "extra-nucellar embryo sac' (Johri et al. 1992: 549) also occurs in Flacourtia (Johri et al. 1992) and approaches the state described for Salicaceae Mirb.where the tip of the embryo sac comes into contact with the integument (Chamberlain 1897;Maheshwari & Roy 1951;Johri et al. 1992).This type of elongated embryo sac may well be characteristic for Salicaceae sensu Chase et al. (2002).Our results on Kiggelaria show that the tip of the embryo sac, clearly indicated by the filiform apparatus of the synergids, remains covered by the nucellus cap (Figure 2C.D) as reported for Achariaceae (Steyn et al. 2001(Steyn et al. , 2002a, b), b).The nucellus cap later forms an epistase.sep arating the zygote and embryo from the micropyle.The presence of an epistase is another clear indication that the tip of the embryo sac stays inside the nucellus.An epistase is only known in a few angiosperm families (Bouman 1984) and was recently reported for Achariaceae (Steyn e ta l. 2001, 2(X)2a).
A detailed comparison of ovule and seed structure in Kiggelaria and Achariaceae (Table 1) shows additional noteworthy similarities, such as the contribution of the nucellus epidermis to nucellus cap formation, sessile ovules with zigzag micropyles (not Ceratiosicyos).suspensorless embryos and sarcotestal seeds.The latter two characters may be important markers for Achariaceae sensu Chase et al. (2002); the embryo of at least Idesia (Flacourtieae) has a long suspensor (Johri et al. 1992).
while sarcotestal seeds were also reported for Caloncoba Gilg and Camptostylus Gilg (Lindackerieae) by Van Heel (1977).In the recently circumscribed Salicaceae the seeds are not sarcotestal.but are often arillate (Chase et al. 2002).
With regard to ovule characters, Kiggelaria seems closer to Acharia and Guthriea than to Ceratiosicyos (Table 1: Nos 3. 5. 6 & 7).Some structural dissimilarities between Kiggelaria and Achariaceae Harms (see Nos 6. 16 & 17) can possibly be linked to specific adaptations for seed dispersal (see Steyn et al. 2002a for details); Kiggelaria seeds are bird-dispersed (Palmer & Pitman 1972) whereas Acharia and Guthriea, but not Ceratio sicyos, are adapted to dispersal by ants.It is noteworthy that seeds of Kiggelaria and Ceratiosicyos, both having relatively large embryos, lack the seed lid found in seeds of Acharia and Guthriea.The absence of such a lid in Kiggelaria supports our hypothesis (Steyn et al. 2002a. b) that this peculiar device is a specific adaptation to seed germination for the small, slow-maturing embryos in seeds of Acharia and Guthriea.
regarded the cyanogenic.rel atively unspecialized flacourtiaceous tribes Berberidopsideae.Erythrospermeae.Pangieae and Oncobeae as closely related.He suggested that phylogenetic relation ships among them and other cyanogenic families of Violales should be examined more carefully.
. C).It is in this position that a fringe layer occurs in the three genera of the Achariaceae (Steyn et al. 2(M)2a.b), but such a layer is absent in Kig gelaria.
observations on Kiggelaria.Seed coat structure in Pangieae, Erythrospermeae and Lindackerieae therefore corresponds to Achariaceae Harms as described by Steyn et al. (2002a, b), providing embryological support for emending the circumscription of Achariaceae as proposed by Chase et al. (2002).