Ovule-to-seed development in Dovyalis caffra ( Salicaceae : Flacour-tieae ) with notes on the taxonomic significance of the extranucellar embryo sac

Dovyalis caffra (Hook.f. & Harv.) Hook.f. is a widespread and horticulturally important southern African endemic. Here described, ovule-to-seed development represents the first embryological information on this genus of the tribe Flacourtieae, Salicaceae sensu lato. Results are discussed in the light of data available on the embryology of the order Malpighiales in general and on the tribe Flacourtieae in particular. It is clear that Dovyalis E.Mey. ex Am. shares many characters with other members of the Flacourtieae. Ovules of D. caffra are anatropous. bitegmic and crassinucellate with both bisporic Alliumand monosporic Polygonum-type embryo sacs. One of the most characteristic embryological features of the tribe is the pres­ ence of an unusual, extranucellar embryo sac that stretches halfway up into the micropyle. Since this extraordinary-shaped embryo sac also defines the embryo sacs of Salix L. and Populus L., it is suggested that the character provides strong sup­ port for the proposed close phylogenetic link between tribes Flacourtieae and Saliceae of Salicaceae sensu lato.

INTRODUCTION Dovyalis E.Mey.ex Am. is a relatively small, mostly African genus comprising about 15 species of shrubs or relatively small trees, often thorny, with simple, alternate and exstipulate leaves (Sleumer 1972).All species are dioecious or rarely polygamous (Langenegger 1976) with a tendency towards andromonoecy, i.e. the male plants occasionally bear a few bisexual flowers and fruits (Sleumer 1972).The apetalous flowers are unobtrusive, greenish, nectariferous and borne singly or in few-flowered fascicles.Fruits are indehiscent, fleshy and edible berries in various species.Dovyalis is widespread in tropical and subtropical Africa (excluding Madagascar), with one species reported from Sri Lanka (Sleumer 1972).Six species occur in the Flora o f southern Africa region (Bredenkamp 2003) where they form a common constituent of mixed scrub, riverine bush, open wood land and forest.
When the genus was first validly described.Amott (1841) regarded the type species, Dovyalis zizyphoides E.Mey.ex Am., as closest to Euphorbiaceae with some resemblance in habit to Flacourtia L'Her.Although Endlicher (1842) consequently listed Dovyalis as a dubi ous genus under his natural order Euphorbiaceae, the association with Flacourtia and a placement of Dovyalis in Flacourtiaceae DC. were subsequently endorsed by most taxonomists during the 19th and 20th centuries (see Warburg 1893 (as Doryalis) \ Gilg 1925;Hutchinson 1967;Lemke 1988).However, the beginning of the 21st century has seen a radical reshuffling of genera tradi tionally placed in Flacourtiaceae.Molecular phylogenet ic studies, supported by data from several other botanical Fields, have indicated that most flacourtiaceous genera fall into two clades, each more closely related to other families within Malpighiales than to one another (Chase et al. 2002).Flacourtiaceae were accordingly split into two groups and the flacourtiaceous genera (not Aphloia DC.) became part of two recircumscribed families, name ly Achariaceae sensu lato and Salicaceae sensu lato.Consequently, all the southern African flacourtiaceous genera were placed in Salicaceae sensu lato with the exception of Kiggelaria L.. Rawsonia Harv.& Sond.and Xylotheca Hochst.; these three genera were included in Achariaceae sensu lato (Chase et al. 2002).
Recent studies on ovule-to-seed development and structure in the three southern African genera of the Achariaceae sensu stricto (Steyn et al. 2001(Steyn et al. , 2002a(Steyn et al. , b. 2003) ) and in Kiggelaria L. (Steyn et al. 2003), offer unequivocal embryological support for the proposed close phylogenetic relationship between the herbaceous, highly modified and monotvpic genera of Achariaceae sensu stricto and the African genus Kiggelaria of the woody tribe Pangieae (Flacourtiaceae sensu stricto).These taxa share a number of uncommon embryological characters, e.g.sessile ovules with zigzag micropyles, deep-lying embryo sacs covered by an epistase in the ovule and seed, endotestal-exotegmic protective layers in the seed coat, suspensorless embryos and sarcotestal seeds with stomata in the epidermis (Steyn et al. 2003).
On the other hand, available embryological and other structural support for emending the circumscription of Salicaceae sensu stricto has been meagre and mainly rests on stamen and pollen characters (Keating 1973;Meeuse 1975;Kaul 1995;Bernhard & Endress 1999).Al though Meeuse (1975) listed some ovule characters shared between Salicaceae sensu stricto and Flacourtiaceae sensu stricto, these characters are not restricted to the two taxa; they are commonly found among related violalean fami lies (Meeuse 1975).Nevertheless, the strong similarity in embryological characters between Achariaceae sensu stricto and Kiggelaria (Steyn et al. 2003) suggests that detailed embryological studies of flacourtiaceous species representing the tribes assigned together with Saliceae Rchb. to Salicaceae sensu lato might also reveal charac ters that are diagnostic for Flacourtiaceae sensu stricto and Salicaceae sensu stricto.Such embryological char acters would be useful for testing hypotheses by molec ular phylogeneticists for a close evolutionary relation ship between these two families.
Here we present data on ovule-to-seed development and structure in Dovyalis caffra (Hook.f.& Harv.)Hook.f., a representative of the tribe Flacourtieae DC. and a species that is, like all the other members of Dovyalis, embryologically completely unknown.Results are com pared with findings recently obtained during a study of seed structure in the polymorphic African willow, Salix mucronata Thunb.(Steyn et al. 2004) and with informa tion found in classic embryological literature on Salica ceae and Flacourtiaceae.Representatives of Flacourtieae are, according to data collected in multidisciplinary stud ies, including molecular phylogeny, possibly the closest relatives of Salix L. and Populus L., both of the tribe Saliceae (Chase et al. 2002).Dovyalis caffra is the only southern African species of the genus that belongs to the section Aberia Hochst., the other five species represent section Dovyalis E.Mey.ex Am. (Sleumer 1972).D. caffra occurs in the easternmost parts of Western Cape and is widespread in the eastern, subtropical regions of southern Africa, extending from Eastern Cape northwards through KwaZulu-Natal, Swazi land, Mpumalanga and Limpopo into Zimbabwe.Com monly known as the Kei apple, D. caffra is also often cul tivated for its edible fruit and, because of its formidable thorns, frequently used as a security hedge.

MATERIAL AND METHODS
Floral buds, open flowers and developing fruits were collected from cultivated female trees of Dovyalis caffra growing in the Pretoria National Botanical Garden and in a private garden in Murrayfield, Pretoria.All flowering stages were dissected to facilitate penetration of chemicals into ovules and seeds and fixed in a 0.1 M cacodylate buffered solution containing 4% formaldehyde and 2.5% glutaraldehyde.Following the methods of Feder & O'Brien (1968), material was dehydrated in an alcohol series and impreg nated with glycol methacrylate (GMA).All material was imbedded in GMA, sectioned transversely or longitudinally at 2-3 pm and subsequently stained with the periodic acid/Schiff reaction and toluidine blue by using the proto cols of O ' Brien & McCully (1981).

Placentation, orientation and development of megagametophyte
The gynoecium in the apetalous female flowers (Figure 1 A) of Dovyalis caffra is usually hexa-or heptacarpellate 102 and very rarely pentacarpellate.The syncarpous, uniloc ular and superior ovary is globose and glabrous (Figure IB) with six or seven styles, each ending in a bifid stig ma.Twelve to fourteen ovules, two per carpel, are borne on parietal placentae developing approximately at the same level, ± halfway down the locule (Figure 1A, B).The ovules occur in pairs on the flanks of the fused carpel margins.These thick structures extend deeply into the locule (Figure 1 A), limiting the space available to the developing ovules so that they show some spatial adjust ment.Nevertheless, longitudinal sections showed that all ovule stages are epitropous with a ventral raphe sensu McClean & Ivimey-Cook (1956: 1392) and anatropous or about to become anatropous (Figure 1C).
The youngest buds examined contained bitegmic, crassinucellate young ovules in the process of curving towards the anatropous position (Figure 1C).The nucellus tissue holds a single megaspore mother cell covered by one or two parietal cell layers.At this early stage the ovule seems to be slightly stalked, but when the curva ture is completed at about the time meiosis takes place, the ovule has no funicle (Figure ID).
Meiosis does not seem to result in a linear tetrad of megaspores.After the first meiotic division, two dyad cells are formed of which the chalazal cell undergoes the second meiotic division, but mostly without the forma tion of a cell wall between the two megaspore nuclei (Figure 2A); in only one ovule a partitioning wall was seen resulting in two chalazal megaspores (Figure 2C).The micropylar dyad cell degenerates without further division (Figure 2B, C).The starting point of the mega gametophyte is therefore usually a bisporic cell with the nuclei situated at opposite poles of the cell (Figure 2B).After two mitotic divisions an eight-nucleate, bisporic embryo sac is formed that conforms to the Allium-type of megagametophyte development (Willemse & Van Went 1984).However, the presence of three megaspores in the nucellus (Figure 2C) suggests that monosporic Poly gonum-type embryo sacs also occur in Dovyalis caffra.

Structure of the mature ovule
Ovules are pendant, sessile, ovoid and anatropous structures reaching a size of about 90 x 60 pm at anthesis (Figure 2D).The two integuments are of equal length so that the micropylar canal is formed by the inner integu ment only, but in post-fertilization stages the outer integu ment usually lengthens to form an exostome (compare Figure 2D, E) that is slightly out of line with the endostome.The lower part of both integuments consists of about five parenchymatic cell layers, but in the micropylar region the integuments become thicker (Figure ID).The outer epidermis of the inner integument is the most promi nent of the integumental layers.The lower part of this epi dermal layer is uniseriate and consists of nieristematic cells with large nuclei and dense protoplasm, whereas the micropylar part is multiseriate and forms most of the cells in the thick micropylar region of this integument (Figure 2D).In the centre of the ovule, the nucellus is an ovoid structure with a slightly attenuate apex consisting of a nucellus cap.The latter is formed by derivatives of the nucellus epidermis and the parietal cell (Figure 2B, C).The nucellus cells are parenchymatic, except at the base of the embryo sac where the cell walls are noticeably thicker, darkly stained and possibly lignified (Figure 2D.E).The cells possibly represent a postament as described by Shamrov (1998: 379), i.e. a column-like tissue located below the sporogenous or gametophytic structures, con sisting of thin-walled or sometimes lignified cells.
The mature embryo sac is a narrow-elliptical structure that occupies an unusual position in the ovule-it lies partly inside and partly outside the nucellar tissue (Figure 2D).In its growth towards maturity, the base of the embryo sac does not succeed in penetrating the cha lazal nucellus, the thick-walled cells of the postament possibly acting as a barrier tissue to stop the encroach ment of the embryo sac into the chalaza (McLean & Ivimey-Cook 1956;Bouman 1984).However, in the micropylar region the apical part of the embryo sac has broken through the nucellus epidermis and.flanked by the inner integument, stretches up halfway into the endostomium (Figure 2D, E).The detailed structure of the embryo sac elements could not be determined.At fer tilization the antipodals have disappeared, the central cell nucleus lies near the egg apparatus in the extranucellar apical region and the synergids show the unmistakable presence of a filiform apparatus (Figure 2E).

Seed development
Embryo: fertilized ovules take about three months to develop into mature, woolly seeds of ± 10x5 mm.embed ded in the fleshy pulp of the fruit, which is a berry.Embryo development was not studied in detail and the embryo could not be typified.The youngest embryo found was ± eight weeks old.Against the wall of the embryo sac the first layers of endosperm cells have been formed.At eight weeks, the embryo is in an early globular stage with a long, uniseriate suspensor stretching up into the micropyle (Figure 3A).The cells of the suspensor contain starch grains and extend past the nucellus epidermis into the region original ly occupied by the extranucellar part of the embryo sac.It seems possible that, by elongating, the suspensor has pushed the embryo proper into the developing endosperm.At seed maturity the embryo is w ithout chlorophyll, erect and spathulate with thin, expanded cotyledons and lies imbedded in oily endosperm.
Seed coat: in Dovyalis caffra the seed coat is not mul tiplicative, i.e. periclinal divisions do not occur during seed development to add extra layers to the seed coat.Since the only mechanical layer in the mature seed coat develops from the outer epidermis of the inner integu ment.the seed is exotegmic.
In the early stages of seed coat development (Figure 3B. D). the testa comprises about four to five cell layers, except in the raphal region where the mesophyll is mul tilayered (Figure 3B).The mesophyll and inner epider mis of the testa consist of thin-walled parenchyma.The outer epidermis of the testa possibly plays an important part in the protection of the seed before the mechanical layer of the tegmen matures.After fertilization, numer ous thin-walled, unicellular epidermal hairs are formed which cover the young seed (Figure 3B.D) at the early globular stage of the embryo (Figure 3A).The hairs grow perpendicular to the seed surface (instead of becoming depressed) and eventually permeate the space between the seeds and the pericarp.The hairs possibly guard against desiccation of the inner seed tissues by prevent ing the loss of water vapour through the numerous stom ata in the epidermis of the testa (Figure 3D).Apart from the hairs, the outer epidermis also contains large num bers of tanniniferous cells with walls impregnated and lumina filled with phenolic compounds.These polyphe nols increase the rigidity of the thin epidermal cell walls and contribute towards the hardness of the seed coat (Werker 1997: 100).Phenolic-containing cells offer re sistance to biodegradation and help to protect against attacks by herbivores, insects and micro-organisms (Boesewinkel & Bouman 1984).In the ripe fruit the outer epidermis with its hairs persists, but the phenolic substances have disappeared and the cells of the meso phyll and inner epidermis collapse (Figure 3C).
At the onset of seed coat development, the tegmen con sists of about five layers (Figure 2E).The outer epidermis of the tegmen (exotegmen) show frequent anticlinal divi sions to form a single layer of thin-walled, radially flat tened cells (Figure 3D) that rapidly stretch in a direction parallel to the longitudinal axis of the seed (Figure 3B).The inner epidermis of the tegmen (endotegmen) also divides anticlinally and forms a layer of small, tightly packed cells containing large amounts of dark-staining phenolic substances (Figure 3B.D).In the ripe fruit the exotegmen forms the mechanical layer of the seed and comprises thick-w alled, radially flattened cells (Figure 3C) w ith cell w alls impregnated w ith phenolic compounds.The mesotegmen has disintegrated and the endotegmen remains as small, strangely formed flask-shaped cells adja cent to the persistent nucellus epidermis that separates the endotegmen from the endosperm (Figure 3C).

DISCUSSION
A detailed comparison of ovule and seed characters in the 36 families placed by Savolainen et al. (2000) in Malpighiales-the clade previously termed Violales (Chase et al. 2002)-is hampered by a lack of compara ble data for many of the families, as given in compendia dealing with comparative embryology.Nevertheless, according to such works (Davis 1966: Corner 1976: Johri et al. 1992;Nandi et al. 1998) similarities include bitegmy.anatropy and crassinucelli with the nucellus epidermis participating in the formation of the nucellus cap (tenuinucellate in Clusiaceae.Dichapetalaceae.Linaceae.Ochnaceae.Trigoniaceae); both integuments usually form the zigzag micropyle canal (inner integu ment only in Dichapetalaceae.Erythroxylaceae.Rhizophoraceae.outer integument only in Salicaceae sensu stricto); a Polygonum-lype embryo sac usually develops, but Allium-and Adoxa-types also occur; endosperm for mation is nuclear, it later becomes cellular and is usually copious (exalbuminous seeds in Caryocaraceae.Clusia ceae.Ochnaceae.Salicaceae sensu stricto); the embryogeny varies considerably, but the mature embryo is typi cally straight, medium-sized to large and protected by a fibrous exotegmen in the seed coat (mesotestal seed in Clusiaceae.exotestal in Dichapetalaceae.Salicaceae).
As far as embryology is concerned.Dovyalis seems to fit comfortably into the framework of Malpighiales (com-   , 3, 4, 6, 7, 8, 9, 10 & 12).Reports in classical literature (Davis 1966;Comer 1976;Johri el al. 1992) indicate that the genus also shares many of the above-mentioned characters with other representatives of the Flacourtieae.e.g.Arechavaletaia uruguayensis Speg., Idesia polycarpa Maxim., Flacourtia indica (Burm.f.) Merr.However, features that seem to stand out as particular to Flacourtieae include the following: an embryo sac that breaks through the nucellus and pro trudes into the endostome.a variation in the functional behaviour of the megaspores and a fibrous exotegmen in the seed (Johri et al. 1992).These are also characters that proved to be definite for Dovyalis in the present study.
Embryological studies on Salix and Populus L. (Chamberlain 1897;Maheshwari & Roy 1951;Nagaraj 1952;Steyn et al. 2004) partly support the placement of Salicaceae sensu stricto in Malpighiales (see Table 1: Nos 1, 2 (partly), 3. 6. 7, 8 (partly).9 (partly), 10 (partly), 11).It is noteworthy that two other embryological characters of diagnostic value for Flacourtieae.namely the unusual extranucellar embryo sac and the inconsistent behaviour of the megaspores, are also characteristic for both Salix and Populus (Chamberlain 1897;Nagaraj 1952).Fur thermore, the hairy seed coat in Dovyalis may be taxonomically significant.Although the seed itself is glabrous in Salix, the genus is characterized by the presence of long, unicellular, intra-ovarian hairs as well as seed with a hairy aril (Steyn et al. 2004).This propensity to produce epidermal hairs in the ovary and its associated structures such as ovules, seed and arils may well reflect a close phylogenetic link between Flacourtieae and Saliceae.Despite all the similarities between Salix and Dovyalis, significant deviations of the basic flacourtiaceous pattern also occur.In Salix the endosperm is ephemeral (Hdkansson 1954).the seed exalbuminous and exotestal and the inner integument absent.Unitegmy results in the lack of an endostome and the absence of a tegmen with its characteristic fibrous exotegmen.Steyn et al. (2004) sug gested that the marked differences in seed coats between Salix and animal-dispersed flacourtiaceous taxa like Dovyalis may possibly be linked to seed adaptations for different dispersal strategies-Salix seeds are mostly dis persed by wind and water, mechanical layers would only unnecessarily increase seed weight, whereas Doxyalis seed occur inside edible berries and need the protection of exotegmic fibres against the onslaught of animal feeders.

TABLE 1 .
-Comparison of selected ovule and seed characters in Dovyalis and Salix pare Table1: Nos 2