The epidermis in Passerina ( Thymelaeaceae ) : structure , function and taxonomic significance

Epidermal features were studied in all 17 species o f Passerina, a genus endemic to southern Africa. Leaves in Passerina are inversely ericoid, the adaxial surface concave and the abaxial surface convex. Leaves are inversely dorsiventral and epistomatic. The adaxial epidermis is villous, with unicellular, uniseriate trichomes and relatively small thin-walled cells, promoting flexibility o f leaf margins owing to turgor changes. In common with many other Thymelaeaceae, abaxial epi­ dermal cells are large and tanniniferous with mucilaginous cell walls. The cuticle is adaxially thin, but abaxially well devel­ oped, probably enabling the leaf to restrict water loss and to tolerate high light intensity and UV-B radiation. Epicuticular waxes, present in all species, comprise both soft and plate waxes. Epidermal structure proves to be taxonomically impor­ tant at family, genus and species levels. Interspecific differences include arrangement o f stomata and presence or absence of abaxial epidermal hair. Other diagnostic characters of the abaxial epidermal cells are arrangement, size and shape, cuticular ornamentation and presence or absence of wax platelets. Two groups of species on the basis o f abaxial epidermal cell orientation are recognised. Many leaf epidermal features in Passerina are interpreted as structural adaptations to the Mediterranean climate of the Cape.


INTRODUCTION
The genus Passerina L. comprises about 17 species, all endemic to southern Africa (Thoday 1924;Bond & Goldblatt 1984).Despite the now outdated revision by Thoday (1924), taxonomic boundaries in Passerina remain a problem, mainly owing to the apparent lack of marked morphological differences between the species.The pre sent paper emanates from a comparative leaf-anatomical survey of the genus, undertaken as part of a monographic study of the group.This survey highlighted the importance of the epidermis as a source of taxonomic evidence.endemic to the Cape Floristic Region.From here the dis tribution of P. filiformis and P. montana extends east and north along the eastern mountains and Great Escarpment of southern Africa.In the Cape the climate is for the most part Mediterranean or semi-Mediterranean.In the west, it rains in winter; along the south coast, winter rainfall is complemented by some summer rain which increases eastwards.The western Karoo and Namaqualand (Suc culent Karoo Biome) are characterised by winter precipi tation and summer drought.KwaZulu-Natal and the east ern mountains of southern Africa are predominantly sum mer rainfall areas.D istribution of the species of Passerina coincides with the geography and climate along the whole distribution area.P. ericoides, P. paleacea, P. paludosa, P. galpinii and P. burchellii are endemic to Western Cape.The first three species are found along beaches and salt marshes only, P. galpinii grows mainly on calcrete in the Agulhas Plain area (Goldblatt & Manning in press) and P. burchellii is found on the high mountains at Genadendal and Villiersdorp.P. comosa grows on mountain slopes and summits in the Kamiesberg, Great Winterhoek and Klein Swartberg Ranges.P. glomerata is found from Worcester to Tulbagh, in the Clanwilliam area and extends to the Witteberg south of Matjiesfontein.P. obtusifolia is ubiqui tous in the Cape, distributed from Worcester in Western Cape to Alice in Eastern Cape and on some of the moun tain ranges in and around the Little Karoo.A new species, of which the plants are often buried under snow during winter, grows at high altitudes in the Ceres Karoo.P. vul garis is a pioneer with a wide distribution from Western Cape to East London in Eastern Cape.P. falcifolia is found on mountain ranges between George and Uitenhage and P. pendula is endemic to the KwaZunga Catchment Basin and the Zwartkops River area of Eastern Cape.P. rubra is common in the Port Elizabeth to Uitenhage area, with outliers in the Swellendam and Bredasdorp Districts.P. drakensbergensis is endemic to the high Drakensberg in the Bergville District of KwaZulu-Natal and P. rigida is distributed all along the coast, from northern KwaZulu-Natal to the Cape Peninsula.Passerina sp.nov. 2 is found on the northern Cederberg Mountains, P. sp. nov. 3 at mountain tops in the Uitenhage area and the Swartberg Pass and P. sp. nov. 4 on the Kouga Mountains and the Montagu Pass.

The combined distribution of all the
The most important studies applying the 'anatomical method' for the delimitation of the Thymelaeaceae were published by Van Tieghem (1893) and Supprian (1894).The presence of mucilaginous epidermal cells in P. eri coides (= Chymococca empetroides Meisn.) as opposed to the total lack thereof in the other species, was also mentioned by Supprian (1894).Subsequently, Gilg (1894) critically discussed the 'anatomical method' as applied by Van Tieghem (1893) and Supprian (1894) for the delimitation of the Thymelaeaceae and concluded that certain characters would not uphold criticism.He regard ed former systems based on floral morphology as more suitable for a taxonomic grouping of the Thymelaeaceae.
The twentieth century yielded very little anatomical work on the Thymelaeaceae.Standard works were those of Solereder (1908) and Metcalfe & Chalk (1950, 1979).Thoday (1921) described the structure and behaviour in drought of the ericoid leaves of P. filiformis and P. cf. falcifolia', he also supplied some notes on their anatomy.In a discussion of inversely dorsiventral leaves, Kugler (1928) included a description of the leaves of P. fili formis (= P. pectinata Hort.).More recently, leaf anato my of the genera Lachnaea L. and Cryptadenia Meisn.was treated by Beyers (1992) and leaf and involucral bract characters of systematic use in Gnidia L. were studied by Beaumont et al. (1994).The scanty informa tion on leaf anatomy in Thymelaeaceae calls for further research in this field, especially in the genus Passerina.
Previous leaf anatomical studies identified mucilagination of the epidermal cells as being of possible taxo nomic importance.Recently Bredenkamp & Van Wyk (1999) clarified the structure of the epidermal cells and origin of the mucilage, concluding that mucilagination of epidermal cells is of taxonomic importance mainly at the family level.
The wide distribution of Passerina in the Cape Floris tic Region, along the southern and eastern coastline and along the Great Escarpment of southern Africa as far north as Zimbabwe, illustrates the adaptation of these plants to a wide range of habitats, including Mediterranean and sum mer rainfall regimes.Decreasing rainfall from the eastern Escarpment to the northwestern Cape is reflected by adap tive changes in the leaf structure of the group.The present paper provides a description of epidermal characters in Passerina as well as an assessment of their taxonomic sig nificance.It also speculates on the possible adaptive value of the observed structural features of the leaf.

MATERIAL AND METHODS
Fresh leaf material of 17 species of Passerina (Table 1) was collected, fixed and stored in a 0.1 M phosphatebuffered solution at pH 7.4, containing 2.5% formalde hyde.0.1% glutaraldehvde and 0.5% caffeine [modified Karnovsky fixative; Kamovsky (1965)].WTienever possi ble, material from at least five different localities was collected, fixed and air-dried for each species and herbarium specimens were made.

Light m icroscope (LM ) studies
The LM was used for general leaf anatomy as well as epidermal studies.Unless stated otherwise, the tenth leaf from the growing point of a twig was used in all com parative studies.To prepare transverse sections of the main vein as well as both leaf margins, a 1 mm wide seg ment of leaf material was cut from the centre of each leaf.Samples were dehydrated, embedded in glycol methacrylate (GMA) and sectioned according to the methods of Feder & O 'Brien (1968).Sections were stained with the periodic acid/Schiff's reaction and in toluidine blue 'O ', then mounted in Entellan (Art.7961, E. Merck.Darmstadt).
The following three methods were followed in the study of the cuticles: 1.
GMA transverse sections of leaves were stained for 10 minutes in 1% Sudan Black B dissolved in 70% ethanol.Sections were rinsed twice in 70% ethanol for a few seconds and mounted in glycerine jelly.2. Cuticular mounts were obtained by maceration according to the method of Kiger (1971).Specimens were slightly over-stained in a 1% acqueous safranin solution, dehydrated in methyl cellusolve and mounted in Entellan.
3. Epidermal mounts were obtained by removing small pieces of ad-and abaxial epidermis manually and by paradermal hand sections.Epidermises were stained in 1% safranin dissolved in 50% ethanol, dehydrated in a graded ethanol series and mounted in Entellan.

Scanning electron m icroscope (SEM ) studies
The SEM was used to study the epidermal surface features (including epicuticular waxes), as well as to ver ify the structure of the cuticle.Leaves from air-dried material were used for all species.Whole leaves were used as they are small and ericoid, but trichomes were manually rem oved adaxially to reveal the stomata.Leaves were mounted onto aluminium stubs with silver paint, exposing the ad-and abaxial surfaces separately and sputter-coated with gold.For the purpose of studying epicuticular waxes, the sputter-coating process was mod ified to prevent high temperatures from changing the wax surfaces.Specimens were sputter-coated for 30 sec onds and left to retain their normal temperature for one minute.This was repeated five times after which the specimens were viewed with a Jeol 840 SEM.
For the verification of the authenticity of epicuticular wax droplets and small round protrusions observed in certain species of Passerina, leaves were washed in chlo roform for one minute, before they were pasted onto alu minium stubs.The procedure described above was used for sputter-coating and viewing.

T ransm ission electron m icroscope (TEM ) studies
The TEM was used for the study of the structure of mucilaginous epidermal cell walls in Passerina.The sec ond, fifth and tenth leaf from the growing points of P. ericoides, P. falcifolia and P. paleacea were used to study the structure of the cell wall.Leaf segments of ± 1 mm2 were fixed in a modified Kamovsky fixative (Karnovsky 1965).Fixed material was rinsed in 0.075 M phosphate buffer, pH 7.4-7.5,post-fixed for one hour in 0.25% aqueous 0 s 0 4, washed in three changes of water, dehydrated in a graded acetone series and embedded in Quetol 651 resin (Van der Merwe & Coetzee 1992).Ultrathin sections were contrasted in 4% aqueous uranyl acetate for 10 minutes and rapidly rinsed in water three times.The sections were then contrasted with lead citrate (Reynolds 1963), rinsed in water and examined with a Phillips 301 TEM.
For the verification of wettability and possible absorption of water by laminar epidermal hairs, we fol low Alvin (1987).He proposed a mechanism through which water is absorbed by the specialised abaxial epi dermal trichomes of Androstachys johnsonii Prain.This process involves the wettability of the hairs which he investigated by spraying the glabrous adaxial surfaces of the leaves with water.Water seeped round the leaf mar gins to the abaxial surface, wetting approximately 50% of the abaxial surface within 5 minutes.In the present study, the glabrous abaxial surfaces of five cymbiform leaves (from dried herbarium specimens) were pasted onto a sticky surface, exposing the villous concave adax ial surface.A drop of water was placed in the adaxial groove at the base of each leaf (average leaf size 2.5 x 4.0 mm) and left overnight.This experiment was repeat ed using 0.5% aqueous safranin, followed after 20 min utes by a rinse with water.

Trichome structure
We have followed the terminology of Stace (1965) and Theobald et al. (1979).

Cuticle
Although the interpretation proposed by Martin & Juniper (1970) for the cuticle of plants has been widely followed by many workers, Holloway (1982) reviewed the historical perspective of the plant cuticle and attempt ed to adopt the most workable interpretation of the cutic ular membrane (CM) in practice.In response, we follow Jeffree (1986), whose uncomplicated and pragmatic inter pretation distinguishes three main zones, namely the cuti cle proper, the cuticular layer and the cell wall.The cutic ular membrane comprises the cuticle proper plus the cuticular layer and is bonded to the outer periclinal walls of the epidermal cells by a pectin-rich layer, which is equivalent to the continuous middle lamella.A layer of epicuticular wax generally coats the cuticle proper.

Cuticular ornamentation (LM and SEM)
We follow Wilkinson (1979) in our choice of termi nology to describe cuticular ornamentation.

Epicuticular wax
The recognition of soft waxes in the present study is based on the criteria proposed by Amelunxen et al. (1967), Wilkinson (1979) and Barthlott et al. (1998).

Cuticle
Transverse section (LM): cuticular membrane 2-5 pm thick, smooth, ridged along boundaries of guard cells (Figure 2G), gradually thickening close to leaf margins, equalling abaxial cuticle in thickness and sculpturing at margins.
Surface view (LM and SEM): smooth (Figure 2C), except in Passerina sp.nov. 1, where markings on epicuticular wax are most probably caused by snow (Figure 3D, E).

Stomatal complex
Transverse section (LM): lamina epistomatic; stoma ta dispersed randomly over adaxial surface, but absent from edges of leaf margin; raised or at same level as other epidermal cells (Figure 2E-H ); dispersed in two columns in adaxial epidermal folds, with ± 3-5 rows of epidermal cells in between; raised, sunken or arranged in stomatal crypts in Passerina sp.nov. 1 (Figure 3F).Guard cell outline in all species varying between widely obtrullate, very widely obtrullate or widely depressed obtrullate, with angles slightly rounded; cell walls thick-  3B) covering outer periclinal walls of epidermal and guard cells, as well as poral epidermal walls of guard cells, smooth or slightly crenate when lining the pore (Figure 3B), contracted into a pair of ± continuous outer stomatal ledges above guard cells, thus forming an entire outer cavity (not divided into compartments); inner stomatal ledges and inner cavity present.Epidermal cells surrounding guard cells not different from other epider mal cells in size, shape or staining properties (Figure 2F).Peristomatal cuticular rims conspicuous on epidermal cells bordering guard cells (Figure 2G).
Wettability and the possible absorption of water by the laminar epidermal hairs in Passerina were assessed by means of laboratory tests.We found that water had formed a film over the felty layer of hair at the leaf base, whereas the adaxial surface had remained dry.A treat ment with 0.5% aqueous safranin revealed that only the exposed parts of the spiralised hairs in the felty indu mentum at the leaf bases stained pinkish.Although the longer hairs at the leaf margins were stained, those on the rest of the adaxial surface remained unstained.

Trichomes
Abaxial surfaces of bracts and young leaves in P. comosa, P. sp. nov. 3 and P. sp. nov. 4 tomentose to sparsely hairy (Figure 4B), older leaves often glabrous.Description of trichomes as described under adaxial epi dermis.

Cuticle
Transverse section (LM): epicuticular wax absent owing to chemical treatment during fixation, embedding and staining.Cuticular membrane (CM) well devel oped.(10-)20-30(-70) fjm thick ( TEM: cuticle structure corresponding to the cuticular structural type 3. described by Holloway (1982).Cuticle proper and CM not distinguishable.Cuticular membrane (Figure 4A) comprising a wide, mainly amorphous outer zone and narrow faintly reticulate inner zone; osmiophilic granules aligned on border of clearly defined cell wall; cuticular pegs with unknown (possibly pectinaceous) substance (stained light grey) between cell wall and peg, forming part of middle lamella.

Cuticular ornamentation
In transections and surface view of leaves, LM and SEM studies showed that two groups of species, hence forth called Groups A. Intermediate and B (Table 3), can be distinguished on the basis of the arrangement and shape of epidermal cells as well as cuticular ornamenta tion.

Group B
Epidermal cells mostly oblong in surface view, arranged in rows; concavities (depressions in centre region of cell) and convexities (roundish cells forming a low dome) more or less alternating (Figure 5G, J); cuti cle with ridges at junction of epidermal cell walls most ly conspicuously raised, exhibiting a definite striate pat tern (Figure 5D.G, J), otherwise ± plane.
Cuticular membrane pronounced at junctions of epi dermal cell walls and grooved between anticlinal walls of adjacent cells (Figure 51), more or less smooth in P. vulgaris.P. filiformis, P. falcifolia.P. pendula, P. rigida.and P. galpinii, except in Passerina sp.nov. 1, in which the presence of snow, at the time of collecting, seemed to have caused markings on the cuticular wax (Figure 5D.E).Small globular papillae visible between cuticular ridges in Passerina sp.nov. 1 (Figure 5D -E 3 and with 9 or 10 globular papillae per cell in P. drak ensbergensis (Figures 5A-C; 6G).

Epicuticular w axes
Soft waxes present, coating entire abaxial surface: wax protruding through amorphous layer of CM in a variety of configurations: droplets conspicuous in P. comosa, P. ericoides and P. burchellii (Figure 6A, D, F); droplets and small round protrusions forming flat, shape less lumps in P. paleacea (Figure 6L).Crystalloids: wax platelets and plates present or absent (Table 3); thin wax platelets, with margins entire or non-entire, flaking from wax surface in P. comosa and P. rigida (Figure 6A.J) and changing to plates as margins become distinctly edged.Upright plates separating from surrounding wax in P. filiformis (Figure 6H).Platelets and plates varying from sparse to abundant: platelets ± square to irregularly shaped, plates ± square to oblong and usually arranged perpendicular to cell rows.
The authenticity of epicuticular wax droplets and small round protrusions, observed in P. ericoides, P. obtusifolia and P. paleacea (Figure 7), was verified by washing leaves in chloroform for one minute and com paring them to unwashed specimens under SEM.Epicuticular wax droplets were clearly discernible in unwashed P. paleacea (Figure 7A), while small pores appeared in the cleaned, de-waxed cuticle after washing (Figure 7B-E).Similar pores were also present in P. eri coides (Figure 7F).No pores were present in the papil late CM of P. obtusifolia, but the corroded apices of the papillae clearly showed an accumulation of epicuticular waxes at these points (Figure 7G-I).

Adaxial epidermis
Plants of high mountains in the tropics usually have straight to curved anticlinal epidermal cell walls, the per centage of species with undulated walls increasing as altitude decreases (Wilkinson 1979).The straight-walled arrangement of the cells in Passerina sp.nov. 1 (Figure 3D.E). a high-altitude montane species, seems to com ply with this pattern.

Stomatal complex
In all but one species ol Passerina the stomata are usu ally raised or at the same level as other epidermal cells (Figure 2E.G. H), indicating that this character is of lim- 100KM U D ited taxonomic value at species level, except in Passerina sp.nov. 1, which has stomatal crypts or sunken stomata.Classification of the stomatal complex into stomatal types is often a problem owing to the subtle distinction of sub sidiary cells (Wilkinson 1979;Van Wyk et al. 1982).Patel (1978) considers subsidiary cells as morphologi cally and physiologically different from other epidermal cells and proposes a number of criteria to distinguish subsidiary cells in mature epidermis.O f these criteria we used the following in the distinction of subsidiary cells: size, shape, contents and position of cells.We found that the cells adjacent to the guard cells did not differ from other epidermal cells, except that they might be raised or sunken (Figures 2K;3C).Furthermore, when stained with PAS, periclinal walls of subsidiary cells should be lightly stained compared with other epidermal cells, owing to less carbohydrates in these cell walls according to Patel (1978).In Passerina the periclinal walls of the cells adjacent to the guard cells stained homogenously with other cells in the stomatal complex (Figure 2F) and the anticlinal walls are not comparatively thinner than those of other epidermal cells, thus the cells adjacent to the guard cells cannot be considered subsidiary cells (Figure 2F, H).Stained with Sudan Black B. the contents of the cells surrounding the guard cells do not differ from those of other epidermal cells and no lipid bodies are pre sent (Figure 2G).
We therefore conclude that the epidermal cells sur rounding the guard cells in Passerina are not differentia ted as subsidiary cells and we classify the stomatal appa ratus in Passerina as anomocytic.This corresponds to the prevailing state in the Thymelaeaceae (Solereder 1908;Metcalfe & Chalk 1979).However, although we prefer to regard the epidermal cells surrounding the guard cells as similar to other epidermal cells, the presence of conspic uous peristomatal cuticular rims on the outer periclinal cell walls of epidermal cells around the guard cells may be used in support of a view that these cells are subsidiary cells.The stomatal apparatus could then be classified as staurocytic (Wilkinson 1979) or anomotetracytic (Dilcher 1974).As the number of epidermal cells surrounding the guard cells varies from 3-5(6), it would seem appropriate to classify the stomatal apparatus as anomostaurocytic (Van Wyk et al. 1982).

Trichom es
Passerina leaves are often cymbiform with spiralised trichomes densely arranged in the concave ventral space.This indumentum is likely to play an important role in the water relations of the plant.Water droplets precipitating from the atmosphere, or running down from leaves directly above, would accumulate in the concave leaf space.Droplets would be repelled by the hydrophobic cuticle of the trichomes and owing to cohe sion forces cause a moisture layer in the upper part of the dense trichomes.One may speculate that water vapour escaping through the stomata would not be drawn outwards by capillary forces because of the water-repelling nature of the cuticle surrounding the tri chomes, thus retaining a high concentration of moisture in the vicinty of the stomata.The overall high concen tration of water vapour over the adaxial surface of the leaf is likely to decrease the transpiration rate.Laboratory tests to assess the wettability and the possi ble absorption of water by the laminar epidermal hairs in Passerina, suggest that the wettability of the spiralised hairs is quite low and that absorption of water by these trichomes is highly improbable.However, our sugges tion of an overall high concentration of water in the adaxial cavity of the leaf, which serves to decrease the transpiration rate, is supported by these tests.

C u ticular o rnam entation
Cuticular thickness may be affected by light, temper ature, soil, atmospheric moisture and altitude (Wilkinson 1979).In Passerina, with many species adapted to the Cape Mediterranean climate, all members have a rela tively thick cuticle, but it was the thickest in P. comosa, P. glomerata, P. burchellii, P. galpinii and P. paleacea (Table 2).The first two species grow in the northwestern parts of the Western Cape and on the mountains in and around the Little Karoo (= Karoo Mountain Centre sensu Weimarck 1941), areas with high light intensity, high temperature and low atmospheric moisture.P. burchellii.growing on high m ountains at V illiersdorp and Genadendal. is exposed to high light intensity as well as high and low critical temperatures.P. galpinii grows on calcrete and P. paleacea is exposed to salt spray and wind.In P. drakensbergensis, P. falcifolia, P. paludosa and P. sp. nov. 1, the thickness of the CM is ± 20 fjm.Of these species, P. falcifolia, from the mountains between George and Uitenhage.and P. drakensbergensis, from high altitudes in the Bergville District of KwaZulu-Natal.are exposed to relatively high atmospheric mois ture.However, it is difficult to speculate on the function al significance of the relatively thin cuticles in P. palu dosa, from salt marshes in the Cape Peninsula, and P. sp. nov. 1, a species from Waboomberg, one of the highest points in the Western Cape and often covered by snow in winter.Habcrlandt (1914), following a study of plants in tropical rain forests, considered the function of papillose epidermal cells as concentrating limited light by acting as lenses.Bredenkamp & Van Wyk (1999) speculate that, in Passerina, the convex outer periclinal epidermal cell wall may well focus light rays onto the mesophyll, whereas large vacuoles filled with phenols and the mucilage formed by the cellulose slimes (inner periclinal walls) protect the mesophyll from potentially dangerous UV-B radiation.According to Wilkinson (1979) 4B -C , G -L; 5A-C).The presence of these papillae could have been induced by the high light inten sity of the areas in which these plants grow.

Epicuticular waxes
In their study of the epicuticular waxes in the families of the Dilleniidae and Rosidae, Ditsch & Barthlott (1997) documented the numbers of genera, species and hybrids in which different wax types occur, without iden tifying the various taxa.The epicuticular waxes of 12 genera, 31 species and two hybrids were studied in the Thymelaeaceae.O f these, nine genera and 26 species have wax flakes, one species has angled platelets and four genera and five species have no crystalloids.Our observations indicate that the simple plate-type waxes found in Passerina correspond well to those described by Ditsch & Barthlott (1997) in the Thymelaeaceae.O f the 17 species in Passerina, two have wax flakes, eight have platelets or angled plates and seven are devoid of crys talloids (Figure 6, Table 3).
The mechanism of wax extrusion through the cuticle is highly controversial (Baker 1974;Jeffree et al. 1975;Hallam 1982).Baker (1982) discusses the extrusion of wax by means of 'pores and channels, the liquid extrusion theory, polymerization theory and the crystallization the ory'.Hallam (1982) proposes that wax or wax precursors in their protein or glycoprotein 'shells' move through the cuticle and burst on the surface, liberating the wax from the "package'; on crystallization, the protein coats stick to the surface as the wax crystals develop.
Our results indicate small pores in the cleaned, de waxed cuticle of P. paleacea and P. ericoides (Figure 7B -F), after washing leaves in chloroform.Both Baker (1982) and Hallam (1982) are convinced that detailed investigations by many investigators have failed to con firm the presence of pores or microchannels in certain plant cuticles and that pores have not been shown to con nect with the plasmalemma of the epidermal cytoplasm below.Although the presence of pores has been con firmed by our study, further research on the ultrastructure of the CM in Passerina could be most informative.
Freeman et al. (1979), working on Citrus, found amorphous wax layers on immature leaves and fruit, with small protrusions and isolated regions of upright platelets developing, eventually followed by cracks and irregular plates.Similarly in Passerina, wax droplets, protrusions and flat, shapeless lumps contribute towards a soft wax coating or a smooth layer.Species of Pas serina with soft wax coatings, without platelets or plates, are summarised in Table 3.In P. comosa, P. friiformis and P. rigida (Figure 6B, H, J) platelets and plates are formed as a result of cracks developing on the outer wax surface, crystallising into irregularly shaped flakes, which gradually become square or oblong with 'entire' or 'non-entire' margins, often becoming distinctly edged.In P.filiform is (Figure 6H) upright plates separate from the surrounding wax, orientating themselves at an angle to the cell rows, eventually resulting in most plates being arranged more or less perpendicularly to the cell rows.Wax type, as well as the presence or absence of plates and platelets, is apparently genetically determined (Baker 1982).For example, P. ericoides, P. rigida and P. paleacea (Figure 6D, J, K) all grow along the sea shore, where they are subjected to wind, salt spray and high light intensity, and yet, P. ericoides and P. paleacea have coverings of soft waxes only, whereas platelets and plates are abundantly present in P. rigida.However, in plate waxes the number of platelets and plates, size, con figuration and distribution of the surface wax structures can be considered as environmentally induced (Baker 1974(Baker , 1982)).

Functions o f epicuticular waxes
Possible functions of epicuticular waxes are discussed by Jeffree (1986).In Passerina, large areas of the abaxi al epidermis are exposed to the atmosphere because the inverse-ericoid leaves are usually closely appressed to the stem.In response to the warm, dry summers of the Mediterranean climate of the Cape, it is proposed that the CM, including the abaxial epicuticular waxes, has a water-proofing function, protecting the leaves against desiccation and limiting transpiration to the adaxial epi dermis only.As the leaves are decussately arranged, the water-repelling function of the waxes would cause droplets of water to run off the abaxial epidermis, into the concave, hairy adaxial surface of the lower leaf, resulting in a decreased transpiration rate owing to the higher adaxial water concentration.According to Jeffree (1986) the wettability of the plant surface is determined by its microroughness.The presence of crystalloid platelets and plates, and especially their arrangement perpendicular to cell rows, may facilitate the retention of moisture.

Systematic value
Epicuticular waxes have been proven taxonomically valuable, among others in the study of the Centrospermae (Engel & Barthlott 1988), Dilleniidae and Rosidae, including the Thym elaeaceae (Ditsch & Barthlott 1997), at sectional level in Eucalyptus L'Her.(Hallam & Chambers 1970) and at species level in Hordeum L. (Baum et al. 1989).In Passerina the pres ence or absence of crystalloid platelets or plates com bined with characteristics of the CM and the outer peri clinal cell walls of the abaxial epidermis, makes it possi ble to distinguish between two groups in the genus.This distinction is species-specific for most of the 17 species examined (Table 3).

Ecological aspects of leaf epidermis
The structure and function of the epidermis should be considered in context with gross leaf morphology and arrangement.Leaf arrangement is of vital importance to the physiology of the plant.The epidermis serves as an envelope, physically protecting the m esophyll, the largest part of the abaxial epidermis forming a multi functional barrier to the environment.The thin adaxial epidermis is concealed in the groove of the cymbiform leaf in most cases; it is almost covered by dense, long, spiralised uniserial trichomes and contains the stomata, which are often raised.This arrangement is likely to reduce the rate of transpiration, especially if moisture can be retained by the indumentum.The abaxial epider mis is probably multifunctional.The whole of the CM has a waterproofing function and the epicuticular waxes also have a water-repelling function.At the same time the CM may play a major part in focusing light rays onto the palisade parenchyma.Large tanniniferous vacuoles may play a role in the possible absorption of UV-B radia tion, and mucilage formed by the cellulose slimes (inner periclinal walls) possibly protects the mesophyll from desiccation (Bredenkamp & Van Wyk 1999).
The expansion and inrolling of the leaf margins in Passerina, as a result of changing turgor pressure in the epidermal cells, were described by Thoday (1921).He regards the main mechanism involved as the co-ordina tion between the turgor pressure and the difference in size and thickness of cell walls of the ad-and abaxial epi dermis, whereas the plicate anticlinal cell walls of the abaxial epidermis protect the cells against bending stress.Stomata (or at least the indumentum) are exposed when the leaf margins expand and are protected in a villous groove when the leaf margins are rolled inwards, thus regulating the rate of transpiration.

CONCLUSIONS
Leaf shape and structure in Thymelaeaceae exhibit a transformation series from mainly dorsiventral, the pre vailing family feature, to isobilateral or centric in Diarthron Turcz., Pimelea Banks & Soland.and Thymelaea Juss.(Metcalfe & Chalk 1950).All the mentioned states are present in Lachnaea and Cryptadenia (Beyers 1992) and, as the most advanced state, inversely dor siventral leaves in Passerina.A transformation series can also be illustrated by the presence of amphistomatic, hypostomatic and epistomatic leaves in the Thyme laeaceae (Metcalfe & Chalk 1950), the epistomatic state in Passerina considered to be the most advanced (the collateral vascular bundles of the leaves, with xylem arranged adaxially and phloem abaxially.rule out the possibility of resupination of the leaves).
The most pronounced epidermal characters of the Thymelaeaceae are anomocytic stomata (Metcalfe & Chalk 1950), unicellular trichomes and mucilagination of epidermal cells.In the present study the presence or absence, distribution of or changes in the above-men tioned structures, were used as distinguishing characters at both generic and species levels.Mucilagination of epi dermal cells is often found both ad-and abaxially in the leaves of Thymelaeaceae.In Passerina, mucilagination takes place in the abaxial epidermis only.At species level the sunken stomata and stomatal crypts of Passerina sp.nov. 1 are used in the delineation of the new taxon and P. comosa is distinguished by the presence of unicellular trichomes on the abaxial surface o f the leaves.
On the basis of abaxial cuticular characters, it has been possible to distinguish two groups of species in the genus.Group A comprises P. burchellii Thoday Hence it can be concluded that the conspicuous dif ferences as well as the concise characters of the ad-and abaxial epidermis, critically described and discussed in this paper, can be used as taxonomic tools at the family.
genus and species levels.Furthermore, the leaf epidermis in Passerina is probably most valuable to the plant in terms of ecological adaptation, considering the wide dis tribution of the genus in southern Africa as well as the accompanying geographical and climatic variation.The gross leaf morphology and the ad-and abaxial epidermal characters have been most useful in the interpretation of the possible functioning of the leaves and are of vital importance in the survival strategies of the plant.

FIGURE 3 .
FIGURE 3.-LM photographs and SEM micrographs in Passerina.A-F.structure of stomatal complex A-C, P ngida.Bredenkamp 1013.Ward 7211 A. surface view of stomata showing penstomatal rims, raised guard cells and pronounced outer stomatal ledges; B. t/s adaxial epider mis stained with Sudan Black B. with crenate surface of cuticular membrane lining poral walls of guard cells; C, epidermal maceration stained with safranin, showing structure of epidermal cells surrounding guard cells, peristomatal nms.D-F, Passerina sp nov. 1, Bredenkamp 1046: D, sunken stomata in cavity of cymbiform leaf; E. epidermal maceration stained with safranin.with structure of epider mal cells and sunken stomata; F. t/s leaf, with raised stomata as well as stomatal crypts.G-J.structure of trichomes G, P rubra, Bredenkamp 905, with poral rims in relation to adaxiaJ epidermal cells H. P. falcifolia, Bredenkamp 915, with unicellular, long, spirahsed.pointed tri chomes; I. P paludosa, Bredenluimp 1035.with tnchome foot and conspicuous lumen.J, P pendula.Bredenkamp 909.tnchomes strongly spiralised.K. L. TEM micrographs of abaxial leaf epidermal cells of P falcifolia.Bredenkamp 917, in cross section: K. mucilage accumu lated between innermost and outermost cellulose layers of inner penclinal cell wall.L. innermost cellulose layer of inner periclinal cell wall Abbreviations: aw, anticlinal cell wall, cy, cytoplasm, up.innermost layer of inner penclinal cell wall; oip.outer layer o f inner penclinal cell wall; m, mucilage; op, outer penclinal cell wall; v, vacuole.Scale bars: K, L 5 pm; A. B. H. 10 pm: C-F, G, I. J. 100 pm ), P. rubra.P. paleacea and P. paludosa (Figure 5J-L).Intermediate Epidermal cells arranged in rows but CM less pro nounced at junctions o f epidermal cell walls and cuticu lar ridges less conspicuous, were recorded in P. comosa (Figures 4B; 6A.B), P. drakensbergensis (Figure 5A.B), P. montana, P. sp. nov. 3 and P. sp. nov. 4. CM smooth or with small globular papillae in P. montana and P. sp. nov.4; domed with a 'm olar'-like crown in P. comosa (Figure 4B, C), with several domes per cell in P. sp. nov.

FIGURE 5 .FIGURE 6 -
FIGURE 5.-Abaxial leaf epidermis and structure of CM in Passerina Epidermal macerations stained with safranin and t/s of epidermis stained with Sudan Black B A -C , P drakensbergensis.Bredenkamp I01H, 1019 A. cells arranged in rows with 9 or 10 globular papillae per cell; B, inner surface facing upwards, cells oblong in shape with 9 or 10 papillae per cell; C, CM layered, with cuticular layer and cuticle prop er, also globular papillae D-F, Passerina sp nov. 1, Bredenkamp 1044, 1046.D, several domes per cell, CM irregularly marked by ice crystals; E, cells arranged in rows, oblong in shape with CM irregularly marked by ice crystals; F, geometrical plates, flat or slightly raised G -I, P. rigida.Bredenkamp 1013.Ward 7211.G, cells arranged in rows, plates abundant.H, cells arranged in rows, isodiametric to slight ly oblong; I, CM pronounced at junctions of epidermal cell walls, grooved in midline of joining walls, concavities and convexities not con spicuous.J-L, P paludosa.Bredenkamp 1035, Thoday 100.J, cells arranged in rows.CM pronounced at junctions of epidermal cell walls, grooved in midline of joining walls, concavities and convexities conspicuous; K. cells arranged in rows, cells oblong; L. CM pronounced at junctions o f epidermal cell walls, grooved in midline o f joining walls.Abbreviations: cl.cuticular layer; co, concavity; cp.cuticle prop er; cw, outer periclinal cell wall; cx, convexity; gr.groove in CM; pa, papillae; pe, cuticular peg; pi.plates Scale bars: A, D, E, G, H. J-L.I(X) pm; B, C, F, 1, 10 pm

Table 2
A the pres ence and prominence of papillae are diagnostically unre liable because they vary with the climate or distribution of the species; only morphologically distinct types can be used for diagnostic purposes.However, distinct epider mal cell papillae characterise P. comosa, P. obtusifolia P. burchellii, P. drakensbergensis and P. sp. nov. 2 (Figures , P. comosa C.H.W right.P. ericoides L.. P. glomerata Thunb.and P. obtusifolia Thoday.Group B comprises P. drakensbergensis Hilliard & B.L.Burtt, P. falcifolia C.H.Wright, P.filiform is L" P. galpinii C.H.Wright, P. montana Thoday, P. paleacea Wikstr., P. paludosa Thoday, P. pendula Eckl.& Zeyh., P. rigida Wikstr., P. rubra C.H.Wright.P. vulgaris Thoday.P. sp. nov. 1. P. sp. nov.2, P. sp. nov. 3 and P. sp. nov. 4. Certain species in each of the two groups seem to be naturally allied.Distribution patterns of P. obtusifolia and P. glomerata coincide at Worcester and transitional types can be clear ly distinguished.Transitional types are similarly present in P.filiform is and P. vulgaris in the Cape Peninsula and in P. ftliformis and P. falcifolia near Knysna.