Ovule and seed structure in Scolopia zeyheri ( Scolopieae ) , with notes on the embryology of Salicaceae

Scolopia zeyheri (N ees) Harv. is a widespread African tree and a member o f the largest genus o f the tropical Old World tribe Scolopieae (Salicaceae sensu lato). This light m icroscopic study is the first report on ovule and seed structure in the genus and the tribe. O vules vary from four to six per ovary, are anatropous. crassinucellate. bitegm ic and occur in an epitropous (rarely pleurotropous). median-parietal position in the unilocular, usually bicarpellate ovary. A very extensive nucellus cap. com prising nucellus epidermal derivatives and parietal tissue, characterizes the young ovule during megasporogenesis and m egagam etogenesis, but the chalazal nucellus is poorly developed. During m eiosis. the micropvlar dyad cell degenerates early. The functional dyad cell forms two m egaspores o f which the chalazal one usually develops into a Polygonum-type embryo sac. At maturity, the micropylar end o f the embryo sac is covered by the remnants o f the nucellus epidermis, the parietal tissue having degenerated. The globular embryo has a short suspensor and lies in nuclear endosperm becom ing cellular. The seed coat develops from both integuments, is tannimferous. has a glabrous surface with stomata and a single layer o f exotegm ic. longitudinal fibres.

Comores, Mascarenes, India and Sri Lanka, SE Asia.Malesia and Australia (Sleumer 1972), Scolopia is the largest and most w idespread genus o f the tribe Scolopieae, Flacourtiaceae sensu auct.This tropical Old World tribe also comprises Hemiscolopia Slooten (1 sp.) from Indomalesia, Mocquerysia Hua (1 sp.) from tropi cal A frica, Phyllobotryon M iill.Arg., including Phylloclinium Baill.(5 spp.) from tropical Africa, and the rather aberrant Pseudoscolopia Gilg (1 sp.) from South Africa (Lemke 1988;Chase et al. 2002).Together with ± 50 other non-cyanogenic genera (and the cyanogenic Banara Aubl.) traditionally classified in  Bernhard & Endress (1999).has yet been given.In fact, apart from data on a few Flacourtieae.Casearia Jacq.(Samydeae) and Oncoha Forssk.(tribal affinities within Salicaceae uncertain), there is a dearth o f embryological informa tion on members o f all flacourtiaceous tribes, currently included in Salicaceae (e.g. Davis 1966;Com er 1976;Van Heel 1977, 1979;John et al. 1992;Nandi et al. 1998).To arrive at a better understanding o f the embry ology o f Salicaceae-an overview o f such characters has not been given for this emended family-and to append the meagre embryological data on its southern African representatives in particular, we here report on ovule and seed characters in Scolopia zeyheri (Nees) Harv.
Commonly known as the thompear or doringpeer (Afrikaans), Scolopia zeyheri is a variable species as regards leaf shape and growth form (Killick 1976

M ATER IA L A N D M ET H O D S
Inflorescences with floral buds, mature flowers, developing fruit and seeds o f Scolopia zeyheri were col lected weekly from trees growing in the Walter Sisulu National Botanical Garden, Roodepoort, South Africa.Inflorescences were immediately immersed and stored in 0.1 M cacodylate buffered solution (pH 7.4) containing 4% formaldehyde and 2.5% glutaraldehyde.Selected ovaries and developing berries were cut from the inflo rescences, sorted according to size and kept in separate, labelled vials containing freshly made up fixative as described above.All stages were dehydrated and impreg nated with glycol methacrylate (GM A) following the methods o f Feder & O 'Brien (1968).Ovules and seeds were dissected from the ovaries and berries, individually embedded in GMA and sectioned sagittally at 2-3 |im.S elected sections w ere stained w ith the periodicacid/Schiff reaction (PAS) and counterstained with toluidine blue, using the protocols o f O ' Brien & McCully (1981).

Placentation, orientation and position o f ovules in the ovary
Scolopia zeyheri bears small, yellowish flowers in inconspicuous, few-flowered axillary racemes at the beginning o f spring (early September in South Africa).The flowers are bisexual, epigynous, nectariferous and sweet-scented.Ovaries are unilocular and bicarpellate (Figure 1 A), less often tricarpellate and develop into red, globular and succulent berries, ± 10 mm diam.Four to six sessile ovules, usually epitropous (micropyle direct ed upwards) with a ventral raphe (Figure 1A) sensu M cLean & Ivimey-Cook (1956: 1392, fig. 1293A), develop in pairs on the fused margins (placentae) o f the carpels, ± halfway down the locule.In a few instances ventral pleurotropous ovules (micropyles directed hori zontally) were found (Figure 1A), indicating that the direction o f the micropyle is not a constant character.The carpel margins extend to some degree into the locule so that, in superficial longitudinal sections o f ovaries, it seems as if the ovary has more than one locule and the epitropous ovules are axile (Figure IB).

Structure o f ovule and development o f embryo sac
Mature ovules are small, ovoid (± 600 x 350 ^m), anatropous, crassinucellate and bitegmic (Figure 2A, B).The chalaza constitutes a small part o f the ovule, there is no funicle and the raphe is unobtrusive.The outer integu ment is longer than the inner and comprises four layers o f isodiametric, thin-walled cells (Figure 2A, B).Large amounts o f dark-staining, phenolic substances occur in the outer epidermis and the mesophyll cells, but the inner epidermis is usually without tannins.The inner integu ment is four-layered at its base and comprises thinwalled, isodiametric cells without tannins.This integu ment becomes thicker in the micropylar region, mostly by periclinal divisions o f the outer epidermal cells (Figure 2A).
The nucellus cylinder is ovoid with a slightly attenu ate apex formed by a few apical nucellus epidermal cells lying opposite the inner opening o f the endostome and separating the apex o f the embryo sac from the micropy le (Figure 2B).The cells are papillate, thin-walled and vacuolate.The base o f the embryo sac is embedded in chalazal nucellus cells with dark-staining contents and walls, representing a postament sensu Shamrov (1998: 379;2002: 135).In Scolopia the first cells o f this struc ture become visible in the short chalazal nucellus region when the megaspores are formed.The postament cells lie directly below the megaspores and in the same longitu dinal row (Figure 3A).It is generally believed that the primary function o f the postament is the transport o f nutrients from the chalaza to the developing megagametophyte (Shamrov 2002).
During meiosis, the micropylar dyad cell does not divide, but degenerates after the first meiotic division (Figure 3A).The functional dyad cell undergoes the sec ond meiotic division, resulting in two megaspores o f unequal size (Figure 3A, B).The chalazal megaspore usually develops into a monosporic, Polygonum-type embryo sac, whereas the micropylar one degenerates (Figure 3B, C).However, sometimes it is the micropylar megaspore that enlarges and functions as the megagametophyte (Figure 3D).No antipodal cells were seen in the chalazal part o f mature embryo sacs, the cells possibly degenerate rapid ly after formation (Figure 4B).The synergids show a dis tinctive filiform apparatus (Figures 2A; 4A) and the egg cell usually protrudes below the synergids.Two separate polar nuclei were often seen near the cells o f the egg apparatus.

Seed coat structure
At maturity, the small, succulent berries contain four to six subovoid, laterally flattened seeds, ± 3.5 x 2 mm with a dark brown, glabrous surface.Both integuments take part in the development o f the seed coat, but neither is multiplicative.In the ripe fruit, the testa consists o f four cell layers on the sides o f the seed and contains large amounts o f tanniniferous substances (Figure 5A -C ).The outer epidermal cells are large, rectangular and thinwalled, except for the outer tangential walls that are thick and, although not lignified, impregnated with tannins that probably help to protect the seed.Scattered stomata occur (Figure 5C).The mesophyll cells o f the testa are small and thin-walled and possibly kept from being crushed by the large amounts o f polyphenols that increase the rigidity o f the cells walls (Werker 1997).The inner epidermis forms a coherent layer o f periclinally flattened cells without tannins.
The tegmen is four to five cells thick and since the outer epidermis forms the main mechanical protective layer, the seed coat can be described as exotegmic.The outer epidermis develops into a compact layer o f thickwalled, lignified (stains blue-green in toluidine blue), pitted and fibre-like sclereids o f about 200-220 ^m long (Figure 5B), stretched in a longitudinal direction.In transverse section the sclereids are ± rectangular (Figure 5A) and show, like the tightly packed, thin-walled meso phyll cells o f the tegmen, no signs o f tanniniferous sub stances.The inner epidermis consists o f small rectangu lar tanniniferous cells, separated from the thin-walled endosperm tissue by shortly periclinally stretched, living cells o f the nucellus epidermis.
The development o f the embryo was not studied; stages found in aborted seeds showed globular embryos with short suspensors (Figure 5D).On account o f their conservative and constant nature (Dahlgren 1991), embryological data have often been successfully employed to test and indicate phylogenetic relationships at different levels o f the taxonomic hierar chy, but most success has prevailed in solving classifica tion problems at the generic level and higher (Stuessy 1990).The present study on Scolopia provides the first detailed information on ovule and seed structure in one o f 21 genera o f an assemblage that putatively forms a natural group among the tribes now included in Salica ceae.This new data allows for a better understanding o f embryological characters in this recently enlarged fami ly.In combination with published information on ovule and seed characters in other genera (Table 1)  t 3 J j g 0 • 1 1 " 5 < j5 £.

3.
Evolutionary status o f Scolopieae.Some characters o f Scolopia suggest a less advanced position for Scolo pieae within the family: (a) At sporogenesis, the megas pore mother cell lies in the weakly developed chalazal nucellus and is covered by a very extensive nucellus cap, mainly derived o f parietal tissue.An extensive apical nucellar region and weakly structured chalaza, regarded as representing an early stage in the evolution o f the dicotyledonous ovule (Shamrov 1998), have not been noted for any other member o f Salicaceae, except in the basal Scyphostegia (Van Heel 1967: 110)  the predictive power of a classification, it is inevitable that so-called paraphyletic groups be recognized, an approach vehemently opposed by some phylogeneticists (Brummit 2002).Variation in embryological features within Salicaceae and Achariaceae displays a heterogeneity not adequately expressed when broadly described at the family level.From evidence gathered thus far, it is clear that embryology has an important role to play in attempts to arrive at a practical classification for the groups traditionally assigned to families such as Achariaceae, Salicaceae, Flacourtiaceae and Kiggelariaceae.For this purpose, informa tion on the embryology of many more taxa is still required.

Flacourtiaceae
, the genera o f Scolopieae were recently placed in a drastically redefined Salicaceae sensu lato (hereafter referred to as Salicaceae), a cosmopolitan family that also include Populus L., SalLx L. and Scyphostegia Stapt'(Chase et al. 2002).Although representatives o f Scolopieae have been known to science since before the beginning o f the 20th century-Scolopia was first described by Von Schreber * South African N ational B iodiversity Institute.Private Bag X I 01, 0001 Pretoria ** H .G.W .J. S c h w eick erd t H erbarium .D epartm ent o f B otany.U niversity o f Pretoria.0 0 0 2 Pretoria.+ A ffiliation: A cock s Chair.Department o f Botany.U niversity o f Pretoria, Phyllobotryon by Muller Argoviensis in 1864-no account o f the tribe's embryology, except for a brief description o f stamen development by FIG U R E 1.-Placentation and orientation o f o v u les in Scolopia zeyheri.A , bicarpellate, unilocular ovary in t/s; B, unilocular ovary in superficial 1/s.a, ovary w all; b, locule; c, fused carpel margins; v, sessile ovu le w ith ventral raphe.N ote pleurotropy (m icropyle directed horizontal ly) in A (left o v u le) and epitropy (m icropyle directed upwards) in B. Scale bars: 500 urn.
During the earliest stages o f development, the m egas pores and young embryo sac (Figures3A -D; 4A) lie deep inside the underdeveloped chalazal nucellus and are covered by a nucellus cap consisting o f an extensive parietal tissue and the derivatives o f the apical nucellus epidermal cells.While the embryo sac matures, the pari etal nucellus tissue is gradually being absorbed by the intrusion o f the micropylar part o f the embryo sac; the chalazal nucellus and most o f the lateral nucellus tissue remain intact.In flowers that have shed their pollen and are on the brink o f fertilization, the micropylar part o f the embryo sac has also absorbed most o f the cells o f the nucellus epidermis.A few remnants o f these papillate cells can still be seen between the wall o f the embryo sac and the inner opening o f the endostomium (Figures2 A; 4B).In S. zeyheri the embryo sac therefore does not extend beyond the limits o f the micropylar nucellus epi dermis to become extranucellar.
now includ ed in Salicaceae (Chase et a i 2002), and compared with findings on Acharia Thunb., Guthriea Bolus, Ceratiosicyos Nees and Kiggelaria L. (Steyn et al. 2003) now included in Achariaceae (Chase et a i 2002), data collec ted during the present study suggest the following: 1. Ovule and seed characters in Salicaceae.Ovules are bitegmic (unitegmic in Saliceae), crassinucellate, anatropous (orthotropous in Oncoba, Casearia Jacq.) and usually sessile or almost so (conspicuous fiinicles in Scyphostegia), occurring on median-parietal, lateralparietal or basal-parietal (basal in Scyphostegia) placen tae in a unilocular, multi-ovular (4-6-ovular in Scolopia) ovary.Integuments are slender (2-5-layered) at the base, both become thicker in the micropylar region and form the straight or slightly skewed micropyle (inner integu ment only in Flacourtia, Oncoba).Although the func tional behaviour o f the megaspores varies, a Polygonumtype embryo sac is often formed, occupying a usually extranucellar position (clearly intranucellar in Scyphostegia andTABLE 1.-Ovule and seed characters in selected genera of S alicaceae.Missing states reflect lack of inform ation Bothalia 35 FIGURE 5.-Seed coat structure in Scolopia zeyheri.A , B, mature seed coat: A , t/s; B , 1/s.C , stom a in seed coat; D , m icropylar region o f seed in 1/s sh o w in g aborted em bryo with short suspensor.i, tegm en (inner integum ent); ii, inner epiderm is o f tegm en; m e, inner openin g o f m icropyle canal; m i, m esophyll o f tegm en; m o. m esophyll o f testa; ne.nucellus epiderm is; o, testa (outer integum ent); oi, outer epiderm is o f tegm en; o o , outer epiderm is o f testa; w, endosperm ; x, stoma; y, em bryo.S cale bars: 100 fxm.
and this may represent a plesiomorphic character within the family, (b) The mature embryo sac o f Scolopia does not attain an extranucellar position as reported for genera in the more advanced tribes o f Salicaceae, nam ely Flacourtieae (Arechavaletaia Speg., Johri et al. 1992; Dovyalis, Steyn et al. 2005; but not Flacourtia, Dathan & Singh 1973), Samydeae (Casearia, Van Heel 1979) and Saliceae (Salix, Chamberlain 1897; Populus, Nagaraj 1952).(c) The embryo in Scolopia has a very short suspensor.Embryos with long, uniseriate suspensors seem to char acterize the advanced tribes Flacourtieae (Idesia Maxim., Johri et al. 1992; Dovyjalis, Steyn et al. 2005), Sam ydeae (Casearia, C om er 1976) and Saliceae (Populus, Nagaraj 1952).The taxonomic significance of the postament in the ovule o f Scolopia is obscure because few workers have recognized this tissue in earli er studies.4. Inclusion o f Scyphostegia in Salicaceae.This monotypic endemic from Borneo, sometimes placed in a family o f its own (Scyphostegiaceae; e.g.Mabberley 1997), shows the presumably plesiomorphic feature o f a distally lobed outer integument, also mentioned for integuments o f Caloncoba Gilg and Camptostylus Gilg (Van Heel 1967, 1977).The latter two genera were recent ly placed in Achariaceae, tribe Lindackerieae (Chase et al. 2002).A nucellus cap o f several layers covering the embryo sac in the mature ovule and persisting in the seed o f Scyphostegia is also reminiscent o f Achariaceae.However, a basal position in Salicaceae (Chase et al. 2002) is supported by the absence o f endotestal sclereids and the presence o f a single layer o f longitudinal endotegmic fibres in the seed coat. 5. Position o/O n co b a in Salicaceae.Presence o f sali coid leaf teeth, absence o f gynocardin-like cyanogenic glucosides (Nandi et al. 1998) and centrifugal stamen initiation (Bernhard & Endress 1999) have previously indicated a position within Salicaceae.M olecular data strongly support the placement o f Oncoba in Salicaceae, whereas the other genera o f erstwhile flacourtiaceous tribe Oncobeae seem well placed in Achariaceae (Chase et al. 2002).The single layer o f exotegmic, longitudinal fibres and destruction o f the nucellus cap cells by the embryo sac (Van Heel 1977) indicate a relationship with salicaceous tribes, but the projecting endostome and orthotropous ovules are somewhat irregular for the family.The dismembering o f the traditional Flacourtiaceae and the recognition o f two major clades that are more closely related to other families in Malpighiales than to one another is one o f the great surprises o f recent molecular phylogenetic analyses (Chase et al. 2002).Undoubtedly many workers familiar with traditional family circumscrip tions viewed these proposals with some scepticism.Subsequently, evidence from em bryology provided strong support for the broad hypothesis o f phylogenetic relationships suggested by Chase et al. (2002), namely the existence o f a Salicaceae sensu lato clade and an Achariaceae sensu lato clade (summarized in Table 1 and also in Steyn et al. 2003).On the other hand, within clades, em bryological features also show variation amongst taxa in at least some cases, most probably reflecting specific patterns o f adaptive radiation, e.g.wind/water-dispersed seed in the tribe Saliceae (Steyn et al. 2004) and myrmechorous seed in Achariaceae (Steyn et al. 2002).How to best translate cladograms into hier archical systems o f classification remains a big chal lenge, considering the different philosophical views on the topic amongst systematists.For example, to enhance H A N , A .S.R .& SIN G H , D. 1973.Structure and developm ent o f o v u le and seed in Flacourtia indica (B u rm .f.) M errill.Proceedings o f the Indian Academy o f Science 39: 172 179.D AVIS, G.L. 1966.Systematic embryology o f the angiosperms.W iley, London.FEDER, N .& O 'B R IE N , T.P. 1968.Plant m icrotechnique: som e prin c ip les and new methods.American Journal o f Botany 55A R A J, M. 1952.Floral m orphology o f Populus deltoides and P. tremuloides.Botanical Gazette 114: 222 243.N A N D I, O .I., C H A SE .M & E N D R E SS. PK 1998.A com bined cladistic analysis o f angiosperm s using rbcL and non-m olecular data sets.Annals o f the Missouri Botanical Garden 85: 1 3 7 -2 1 2 .O 'BR IEN .T.P. & MCCULLY, M E. 1981.The stuihofplant structure: principles and selected methods.Termacarpi, M elbourne.PALM ER.E. & PIT M A N , N. 1972.Trees o f southern Africa, vol.