Leaf anatomy of some southern African Pavetta species

The anatomy of the leaf blade and petiole of 16 indigenous southern African Pavetta taxa was studied. The ordinary epidermal cells, stomata, non-glandular hairs, mesophyll and main veins are described. Particular attention was paid to features with a potential taxonomic value. The relationship between the families Rubiaceae, Oleaceae and Oliniaceae is discussed. S. Afr. J. Bot. 1986, 52: 489-500 Die anatomie van die blaarlamina en petiool van 16 inheemse Suider-Afrikaanse Pavetta-taksons is bestudeer. Die gewone epidermisselle, stomas, nie-klierhare, mesofil en hoofare word beskryf. Besondere aandag is aan kenmerke met 'n potensiele taksonomiese waarde geskenk. Die verwantskappe tussen die families Rubiaceae, Oleaceae en Oliniaceae word bespreek. S.-Afr. Tydskr. Plantk. 1986, 52: 489-500


Introduction
Native southern African Pavetta species usually have simple, decussate leaves with entire leaf margins. Sometimes there are three leaves per whorl and some taxa have hairy leaves. The leaves are distinctly petiolated or subsessile. Pavetta species have in their leaves bacterial nodules which are visible as black spots when the leaves are viewed against the light. There is considerable information available on the morphology of the bacterial nodules in the leaves but very little is known about the anatomy of the leaves of Pavetta species. As physiological studies have been undertaken on the bacterial nodules (Grobbelaar eta/. 1971;Grobbelaar & Groenewald 1974) and a taxonomic revision of the southern African members of the genus has been completed (Kok & Grobbelaar 1984), this anatomical study was undertaken to contribute further towards the overall understanding of the genus Pavetta in southern Africa.

Materials and Methods
A list of specimens studied is given in Herman et a/. (1986). Additional material studied is listed in Table I. Voucher specimens are housed in the National Herbarium, Pretoria (PRE) and/or the Schweickerdt Herbarium, Botany Department, University of Pretoria (PRU).
Material was prepared as previously described (Herman et a/. 1986). Transverse sections were made of the proximal, median and distal parts of the petiole to study the course of the vascular tissue. Transverse sections were also made of the median part of the lamina. Epidermal segments prepared according to the method of Dilcher (1974) were obtained from both leaf surfaces opposite the midvein, the intercostal area and the leaf margin. The epidermal preparations were stained

490
with safranin and mounted in Canada balsam. Transverse sections of fresh leaves were stained with Sudan 4 to test for the presence of lipids in the mesophyll.
The surfaces of the leaves , obtained from herbarium specimens, were studied with a Hitachi-Akashi MSM-4 scanning electron microscope after being coated with gold.
The usage of the term taxonomic importance in this article refers to the diagnostic value of certain characters, that is, to distinguish between taxa, except in the discussion of the anatomy of the petiole where it is used for the phylogenetic value, namely to determine relationships between taxa.

Results
The petiole S.-Afr. Tydskr. Plantk., 1986, 52(6) The outline of the petiole of P. cooperi, P. barbertonensis and P. natalensis is terete whereas in the rest of the species studied it is semi-terete with the adaxial surface flattened and slightly to significantly winged.
In transverse section the epidermal cells appear round to square but are papillate in P. inandensis and P. lanceolata. A few stomata were observed in the epidermis. Uni-and bicellular non-glandular hairs occur in the epidermis of P. cooperi, P. barbertonensis, P. gracilifolia, P. capensis subsp . komghensis, P. schumanniana and P. gardeniijolia var. subtomentosa (Figure 1). The bicellular hairs consist of a basal cell surrounded by raised neighbouring cells and an acute terminal cell. In all the specimens studied, a median and two or more lateral, adaxially located vascular bundles were observed (Figure 1). The petiolar ground tissue consists of peripheral 491 lacunar to angular collenchyma with thin-walled parenchyma around the median vascular bundle (Figures 1 & 2). Crystal sand often occurs in this ground tissue. The median vascular bundle is surrounded by a well-defined starch sheath ( Figure 3).
The main vascular bundle is collateral. The phloem is distinct and primary phloem fibres often occur except in P. barbertonensis, P. gracilifolia and P. revoluta. The number of radial rows of vessel elements in the main vascular bundles appear to be a feature with potential taxonomic value.
The leaf blade Epidermis Ordinary epidermal cells. The ordinary epidermal cells are square to rectangular as seen in a transverse section. The adaxial epidermal cells are usually larger than the abaxial cells (measured perpendicular to the leaf surface in transverse section) ( Figure 6). The adaxial epidermal cells of P. catophylla are markedly larger (42-45 J.lm) than those of the rest of the investigated species (< 38 J.lm).
In surface view, under the light microscope, the intercostal epidermal cells are polygonal with straight walls (Figure 7) or they have slight to conspicuously sinuous walls ( Figure 8). When both the ab-and adaxial intercostal cells have sinuous walls, those of the abaxial epidermis are generally more sinuous than those of the adaxial epidermis ( Table 2). The abaxial costal epidermal cells are brick-shaped with straight walls and arranged in files parallel to the vein ( Figure 9). The adaxial costal epidermal cells are similar in shape to those of the abaxial epidermis, in the case of the larger veins only. The epidermal cells of the leaf margin are also brick-shaped with straight walls and arranged with their long axes parallel to the long axis of the blade. Viewed under the SEM, it can be seen that the leaf margins of P. lanceolata are papillate (Figure 1 0) in contrast to the leaf margins of the other taxa ( Figure 11) which are smooth.
Stomata. The stomata of all the species investigated are paracytic ( Figure 8). The leaves of P. edentula and P. zeyheri are amphistomatic whereas the leaves of all the other species studied are hypostomatic. In taxa where the intercostal Sin.
S.-Afr. Tydskr. Plantk., 1986, 52(6) epidermal cells have sinuous walls, the walls of the subsidiary cells are not sinuous or are only slightly so. An SEM study revealed radiating striae in the cuticle around the stomatal pores of P. cooperi, P. barbertonensis and P. gardeniifolia ( Figure 12).
Non-glandular hairs. Non-glandular hairs occur on both the adaxial and abaxial surfaces of the leaves of P. cooperi, P. barbertonenis, P. gracilijolia, P. capensis subsp. komghensis, P. schumanniana, and P. gardeniifolia var. subtomentosa. Generally the indumentum on the main and secondary veins is more dense than in the intercostal areas.
The indumentum consists of uni-and bicellular non-glandular hairs. The bicellular hairs consist of a basal cell and an acute terminal cell ( Figure 13). Spiral thickenings are visible in the cell walls ( Figure 14). An ultrastructural study showed that protuberances occur on the hairs (Figures 15 & 16). In taxa where the epidermal cells have sinuous walls, the epidermal cells adjoining the hairs do not have sinuous walls or at most have cell walls that are only slightly . sinuous (Figure 8).

The mesophyll
The leaves are dorsiventral ( Figure 17). The palisade parenchyma is one-or two-layered in P. inandensis, P. lanceolata, P. kotzei, P. cooperi, P. catophylla, P. barbertonensis, P. gracilifolia, P. natalensis, P. capensis subsp. komghensis, P. schumanniana, P. gardeniifolia and P. zeyheri and threeto four-layered .in P. revoluta, P. capensis subsp. capensis and P. edentula. In P. revoluta, P. natalensis and P. capensis subsp. capensis there is no distinct zonation of the palisade and spongy parenchyma but a gradual transition occurs from one tissue into the other. The palisade parenchyma cells of P. barbertonensis are short and broad and can only be distinguished from the spongy parenchyma on account of their compact arrangement. The amphistomatic leaves of P. edentula and P. zeyheri contain substomatal chambers and relatively large intercellular spaces in the palisade parenchyma. Spherical bodies, identified as oil drops using Sudan 4, occur in the mesophyll cells of fresh P. edentula leaves. Collenchyma cells were observed in the leaf margin ( Figure 18) and crystal sand in the mesophyll tissue. The intercostal dorsiventral thickness of the leaf blade taken 186 J.lm from the main vein ( Figure 6), appears to be a feature with potential taxonomic value. Of the species studied, P. barbertonensis has the thinnest leaves (13-16 J.lm).

The main vein
Apart from P. catophylla, P. gracilifolia, P. natalensis, P. capensis subsp. capensis and P. zeyheri, the adaxial surface of the blade opposite the main vein is conspicuously raised (Figure 19). In transverse section the main vein of P. cooperi, P. catophylla, P. revoluta, P. natalensis, P. edentula, P. schumanniana and P. gardeniifolia is circular (Figure 20), whereas in the remaining species it is crescent-shaped with or without additional adaxial vascular bundles ( Figure 19). The additional bundles develop where branching occurs and gaps appear in the vascular cylinder. The vascular bundles are collateral as in the petiole. The number of radial rows of vessel elements in the main veins appear to be a feature with potential taxonomic value. Phloem fibres were observed in the phloem of all the taxa except P. barbertonensis and P. gracilijolia.
The main veins are ensheathed by thin-walled, isodiametric parenchyma cells which, however, do not constitute a distinct bundle sheath. This thin-walled tissue often contains crystal sand. Between the thin-walled tissue and the abaxial and adaxial epidermis a few layers of collenchyma cells are present ( Figure 19). Collenchyma occurs adaxially and abaxially of the secondary veins as well, but it is less pronounced adaxially S.-Afr. Tydskr. Plantk., 1986, 52(6) in P. inandensis, P. /anceo/ata, P. kotzei, P. capensis subsp. capensis and P. zeyheri. The thickness of the blade across the main vein (Figure 6), as well as the ratio: thickness across  the vein to thickness across the leaf blade, appears to be another feature with potential taxonomic value.

Bacterial nodules
The mature bacterial nodules usually extend across the thickness of the blade and sometimes cause the adaxial surface to be raised. In transverse section the nodules are round or oval and are often associated with the main or secondary veins ( Figure 21). Above the nodule there is a tube (Figure 22) which can be seen as a pore in the adaxial epidermis ( Figure 23). This tube develops as a result of the adaxial epidermis being indented above the bacterial nodule, ending in a sunken stoma which is already present in the leaf primordium (Herman et a/. 1986). The bacterial nodule is surrounded by a sheath of two to three layers of thin-walled, elongated parenchyma cells ( Figure 24). The nodule is traversed by similar cells dividing it into chambers in which the bacteria are located (Figure 24).

The petiole
The petiole anatomy appears to be of greater taxonomic importance than is generally accepted, provided it is used with discretion (Hare 1942-3). From this study it is clear that the main vascular bundles of the Pavetta species under consideration fall into two types as far as their shape is concerned, namely cylindrical and crescent-shaped. The cylindrical main vascular bundle is mainly found in taxa with petiolate leaves e.g. P. kotzei, P. cooperi, P. barbertonensis, P. revoluta, P. natalensis, P. capensis, P. edentu/a, P. schumanniana and P. gardeniijolia. A crescent-shaped main vascular bundle, on the other hand, is found in the subsessile leaves of P. lanceo/ata and P. gracilijolia. The main vascular bundle leaves the leaf gap as a collateral bundle and therefore it can be postulated that it needs a minimum length of petiole to expand, curve into the crescentshape and eventually fuse adaxially into a cylinder. As the main vascular bundle of all the taxa studied is crescent-shaped at the base of the petiole (Herman eta/. 1986), it is possible that in those species with very short petioles, the cylindrical type could not develop fully. There are exceptions however: P. catophy/la and P. zeyheri both have a cylindrical main vascular bundle but have leaves with short petioles while P. inandensis has a crescent-shaped main vascular bundle and leaves with long petioles. These species are not closely related to each other (Bremekamp 1934). It is thus difficult to draw a phylogenetic line for the taxa studied. However, when the shapes of the main vascular bundles are related to Bremekamp's (1934) classification, the following pattern emerges: P. inandensis, P. lanceolata and P. kotzei are classified in the section Aethiopinymphe. Within this section a possible line of development can be seen: P. kotzei has leaves with long petioles and a cylindrical main vascular bundle in the petiole; P. inandensis has long petioles and a crescent-shaped main vascular bundle, whereas P. lanceolata has subsessile leaves with crescent-shaped main vascular bundles. Likewise in the section Crinita there are leaves with long petioles and a cylindrical main vascular bundle in P. cooperi, P. barbertonensis, P. revoluta, P. natalensis and P. capensis, subsessile leaves in P. catophy/la with a cylindrical type and subsessile leaves in P. gracilijolia with crescent-shaped main vascular bundles. Stebbins (1974) proposed the hypothesis that the sessile leaves of the original Anthophyta had elliptic or obovate laminae which gradually formed an indistinct petiole. The leaves of early dicotyledonous plants developed quickly from this form into leaves with a distinct petiole and leaf blade. S. -Afr. Tydskr. Plantk. , 1986, 52(6) Applying this hypothesis to the length of the petiole and shape of its main vascular bundles in the Pavetta species studied, the following can be deduced: in the section Aethiopinymphe, P. kotzei is probably the most advanced species with a long petiole and a cylindrical main vascular bundle. P. inandensis with its long petiole but crescent-shaped main vascular bundle then represents a possible intermediate evolutionary form while P. lanceolata with its short petioles and crescent-shaped main vascular bundles would represent the most primitive form. Likewise in the section Crinita, P. cooperi, P. barbertonensis, P. revoluta, P. natalensis and P. capensis with long petioles and a cylindrical main vascular bundle are possibly the most advanced species. P. catophy/la with short petioles and a cylindrical main vascular bundle represents a possible intermediate form with P. gracilijolia with its short petioles and crescent -shaped main vascular bundles being the most primitive form. It should be borne in mind, however, that only 16 taxa out of a genus of about 406 species have been studied. This method of identifying a possible phylogenetic line could, nevertheless, be used as a guide to the rest of the genus. Kiew & Ibrahim (1982) studied Chionanthus and Olea anatomy (Oleaceae) and it seems as if the petiole anatomy of these species resembles that of the Pavetta species studied, as far as the shape of the petiole and main vascular bundles of the petiole and the general anatomy is concerned. Dahlgren (1975) grouped the order Oleales and the order Gentianales, containing the family Rubiaceae, in the superorder Gentiananae, thereby showing a close relationship between the families Oleaceae and Rubiaceae. This relationship is supported by the similarities in the petiole anatomy of Olea, Chionanthus and Pavetta. Mujica & Cutler (1974) described the anatomy of 0/inia species (Oliniaceae), including South African representatives. Here again similarities exist in the petiole anatomy of Olinia and Pavetta and both these genera possess paracytic stomata. This serves as an indication of a possible relationship between the families Oliniaceae and Rubiaceae.

The leaf blade
The anatomy of the leaves reflects to some extent the environmental conditions where the species grow. Most of the Pavetta species studied are adapted to xerophytic conditions as was also indicated by Schumann (1891). The leaf blades of P. revoluta, a coastal shrub, and P. capensis subsp. capensis are thick, shiny, leathery and with a thick cuticle. The large adaxial epidermal cells of P. catophylla are also considered to be an adaptation to xerophytic conditions (Roth 1980). The pronounced unilayered palisade parenchyma found in most Pavetta species studied, as well as the presence of non-glandular hairs covering the leaves of some species, are also indicative of an adaptation to xerophytic conditions (Schimper 1903;Bews 1925;Shields 1950;Uphof eta/. 1962;Fahn 1964;Roth 1980). In contrast, the palisade parenchyma cells of P. barbertonensis are short and broad. This species grows under dense shady conditions and Cutter (1971), Metcalfe & Chalk (1979) and Roth (1980) have described these short, broad cells in shade leaves. Although the features described are ecological adaptions, they can be used as taxonomic criteria. For instance, the presence or absence of non-glandular hairs can be used to divide the southern African species of the genus into two groups (Herman 1983); the large adaxial epidermal cells of P. catophylla serve partially to distinguish it from other species with similar morphology and the short, broad palisade parenchyma cells of P. barbertonensis result in the leaves being very thin, which makes it an excellent taxonomic criterion for use in the field and the herbarium. Bremekamp (1929) considered the revolute leaf margin to be a generic feature. Although this is obvious in herbarium material, it is not always visible in live and preserved material. This artificial characteristic is caused by the fact that there are collenchyma cells in the leaf margin. The cellulose cell walls of these cells are saturated with water and when the plant loses water during the process of drying, the cell walls shrink and the leaf margin rolls abaxially to form a revolute leaf margin. Lersten & Curtis (1977) observed spiral wall thickenings in trichomes in the shoot apices of P. lasiopeplus, similar to those observed in the non-glandular hairs on the leaves of the Pavetta taxa studied. Uphof et a!. (1962) quoted several authors describing these spiral wall thickenings in trichomes, especially in Gossypium. Pant & Mehra (1965) studied the ontogeny of stomata in some Rubiaceae. The general structure of the epidermal cell walls and stomata of the southern African Pavetta species agrees with that described by these two authors. According to them, amphistomatic leaves are known in the family Rubiaceae. The amphistomatic leaves of P. edentula and P. zeyheri are, therefore, not an exception for the family Rubiaceae but this feature can be used as a taxonomic criterion in the genus Pavetta. Zimmermann (1902), Von Faber (1912, Boodle (1923), Humm (1944), Lersten & Horner (1967), Whitmoyer & Horner (1970), Van Hove (1972) and Lersten & Horner (1976) have described the development and morphology of the bacterial nodules in various representatives of the family Rubiaceae. The development of the bacterial nodules in Pavetta was not examined in this study. The morphology of the bacterial nodules agrees with that described by the above authors. In most of the relevant literature prior to 1970, it was claimed that the bacteria in the bacterial nodules were capable of fixing free nitrogen (Zimmermann 1902;Von Faber 1912;Boodle 1923;Silver & Centifanto 1963;Centifanto & Silver 1964;Grobbelaar et at. 1971). The majority of the contemporary research does not agree with this view (Seeking 1971;Silvester & Astridge 1971;Grobbelaar & Groenewald 1974). Several studies have shown that dwarf plants, ('cripples'), develop from Psychotria seed which have been treated with hot water in order to produce plants that are free of bacterial leaf nodules (Humm 1944;Scott 1%9;Becking 1971). The possibility exists that the bacterial symbiont produces a growth stimulant or growth regulator which is necessary for the 'normal' growth of the plants (Humm 1944;Silver & Centifanto 1963;Bond 1967;Scott 1969;Becking 1971;Grobbelaar 1971;Van Hove 1972;Edwards & La Motte 1975;Lersten & Horner 1976). This aspect is still under investigation (Herman 1983).

General
The general anatomy of the leaf of the Pavetta species studied agrees with that described for the Rubiaceae by Solereder (1908) and Metcalfe & Chalk (1950). Anatomical features of the petiole which appear to be of diagnostic value, are the shape of the main vascular bundles, the number of radial rows of vessel elements in the main vascular bundles and the length of the petiole. Diagnostic anatomical features of the leaf blade which appear to be of taxonomic value, are: (l) leaf blades amphi-or hypostomatic (2) cuticle striated around stomata or smooth (3) leaf margin papillate or smooth (4) number of palisade parenchyma layers (5) shape of the main vein of the blade 499 (6) the number of radial rows of vessel elements in the main vein of the leaf blade (7) the thickness of the leaf blade across the main vein (8) the thickness of the intercostal regions of the leaf blade (9) the ratio of the dimensions in 7 and 8 above (10) non-glandular hairs present or absent These features together with the external morphology of the leaves have been used in a numerical analysis. The external morphology of the leaves and the results of the numerical analysis will be described in a separate publication.