The morphology of the vegetative shoot apex and stem of some southern African Pavetta species

The morphology and anatomy of the vegetative shoot apex, internode and node of 16 indigenous southern African Pavetta species were studied. The glandular hairs occurring in the shoot apex are described and the presence of fully developed stomata, observed in the shoot apex, pointed out. A periderm is formed in the cortex of the stem. Crystal idioblasts occur among the primary phloem fibres and sometimes in the pith of the stem. The node is unilacunar. Branches from the leaf trace supply the stipules with vascular tissue.


Introduction
The genus Pavetta L. belongs to the family Rubiaceae and the species are either trees, shrubs or dwarf shrubs. In southern Africa they are restricted to the summer rainfall area where some of them are endemics (Bremekamp 1929;Palmer & Pitman 1972). They are commonly known as bride's bushes and have bacterial leaf nodules. Two species, viz P. harborii S. Moore and P. schumanniana F. Hoffm. ex K. Schum., are said to cause the disease known as gousiekte in domesticated ruminants (Codd 1961;Codd & Voorendyk 1966;Vahrmeijer 1981).
The taxonomy of the genus is poorly known. The only taxonomic work on the genus Pavetta in southern Africa consists of a revision and a monograph of the genus by Bremekamp in 1929 and1934 respectively. A revision is currently being undertaken by Kok & Grobbelaar (1984).
Very little has been published on the morphology of the genus . Information on the general morphology of the family Rubiaceae can be found in Solereder (1908) and Metcalfe & Chalk (1950). Specific information on the structure of glandular trichomes in the shoot apex, bacterial nodules in the leaves, wood anatomy and node structure is also available but mostly for other genera in the family .
The aim of this study was to examine the anatomy of the vegetative shoot apex and young stem in order to contribute to the taxonomic study presently underway (Kok & Grobbelaar 1984). The morphology of the mature leaves will be described in a separate publication.

Materials and Methods
Sixteen indigenous taxa of the genus Pavetta were studied.
Specimens and herbarium vouchers used in this study are listed in Table 1. Voucher specimens are kept in the H.G.W .J . Schweickerdt Herbarium (PRU), University of Pretoria and/ or the National Herbarium (PRE), Botanical Research Institute, Pretoria.
Young stems were fixed in FAA in the field . In most cases specimens from three localities were examined. Fixed material was mainly used but in a few cases where fresh material was not available, rehydrated herbarium material was used (Herman 1983).
Material was embedded in paraplast wax (Johansen 1940) Table 1 List of voucher specimens. Species arranged according to Bremekamp (1934). Names according to Kok & Grobbelaar (1984)   and cryo sections were also prepared from stem material. Wax sections were stained with safranin and fast green (Johansen 1940) and mounted in canada balsem. The 'Periodic acid -Schiff Reaction' (PAS) was carried out on GMA sections following the method of Feder & O'Brien (1968), using 0,50Jo 2,4 dinitrophenyl hydrazine (DNPH) in 1507o acetic acid (for 30 min) as the blocking agent. Sections were counterstained for 5 min in 0,050Jo toluidine blue. Some sections were stained with toluidine blue only. All GMA sections were mounted in either Parmount or Entellan.
Other GMA sections were used for: (a) staining for lipids in a saturated solution of Sudan Black B in 7007o ethanol, for 10 min (Van der Schijff & Robbertse 1976). After a rinse in 7007o ethanol, they were mounted in liquefied glycerine jelly; (b) staining lignified cell walls by mounting them in a drop of saturated phloroglucinol in 2007o HCI.
Hand and cryo sections were stained with toluidine blue and mounted in liquefied glycerine jelly.
Macerates were prepared by carefully heating test tubes containing pieces of the young stems submersed in Schulze's solution (McClean & Cook 1941) in a water bath. The macerated material was thoroughly washed with water, stained with safranin and mounted in liquefied glycerine jelly.

Results
The vegetative shoot apex The vegetative shoot apex is conical in comparison with the reproductive shoot apex which is inverted cyathiform. The apical dome is covered by successive layers of stipules and leaf primordia ( Figure 1). The outer layer represents the fused interpetiolar stipules of the youngest pair of aborted leaves.
Non-glandular hairs are present on the abaxial surface of the stipules of P. cooperi, P. barbertonensis, P. gracilijolia, P. capensis subsp. komghensis, P. schumanniana and P. gardeniifolia var. subtomentosa. The adaxial surface of the stipules of the species studied are also covered with nonglandular hairs ( Figure 2) although very few or no hairs were observed in P. inandensis, P. lanceolata, P. kotzei, P. barbertonensis, P. edentula and P. schumanniana. Glandular hairs, which are known as dendroid.colleters (Lersten 1974b), occur on the adaxial surface of the stipules of all the species ( Figure  3). These dendroid colleters consist of a multicellular axis bearing elongated branch cells which are distally entirely separated from each other (Figures 4 & 5). A histochemical test using Sudan Black B demonstrated a very distinct cuticle on the outer colleter cell walls ( Figure 6).
The leaf primordia consist of meristematic tissue ( Figure  7) whilst the protoderm cells are cubical. At this stage, no stomata are visible in the abaxial protoderm though fully developed sunken guard cells occur in the adaxial protoderm ( Figure 8).
In longitudinal section the promeristem is flat to slightly dome-shaped. The promeristem can be divided into an outer two-layered tunica and a central multi-layered corpus ( Figure  10). The tunica cells are cubical and contain dense granular material while the corpus cells are isodiametrical. The contents

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. ,~· of the corpus cells appear to consist of less dense material. The tissues of the shoot apex often contain crystal sand.
The dendroid colleters occurring on the adaxial surfaces of the stipules have completely disintegrated by the time the young stem grows through the stipules covering the vegetative shoot apex.

The internode
The internode is round to oval in transverse section. The epidermal cells are spherical to cubical except for P. inandensis and P. lanceolata which have papillate epidermal cells. Unior bicellular trichomes occur in the epidermis of P. cooperi, P. barbertonensis, P. gracilifolia, P. capensis subsp. komghensis, P. schumanniana and P. gardeniijolia var. subtomentosa.
In transverse section the cortex appears to consist of circular parenchyma cells (Figure 11) of which the peripheral cells are sometimes differentiated into lacunar collenchyma -a term suggested for this type of tissue by Esau (1960) and Carlquist (1961). A well defined starch sheath encircles the stele. A periderm is formed at an early stage in the cortex (Figure 11). The histochemical test with Sudan Black B showed that the radial and outer tangential walls of the first phellem cells are rich in lipid (Figures 12 & 13). In one specimen of P. inandensis (Herman 54) three consecutive periderms were observed.
It is difficult to distinguish between the individual collateral vascular bundles of the stele because secondary growth commences at an early stage ( Figure 11). The phloem contains variable amounts of primary phloem fibres as well as crystal idioblasts, called crystal sclerenchyma by Solereder (1908). This study showed that secondary layers are formed in the radial and inner tangential cell walls of the crystal sclerenchyma (Figures 14 & 15). A phloroglucinol-HCl test showed that these secondary cell walls are lignified. The crystals contained in: the crystal sclerenchyma are rhomboidal.
The xylem vessels are mostly arranged in radial multiples or groups and are occasionally accompanied by tracheids. The occurrence of tracheids was confirmed by examining the macerates.
The pith consists of thin-walled parenchyma cells which are circular in transverse section. Some of these cells develop thick walls during maturation to form crystal sclerenchyma as in the phloem (Figure 16). Crystal sand also occurs in the thinwalled tissues of the cortex and pith.

The node
Pavetta species have decussate leaves with interpetiolar stipules. The stipules of each individual leaf are fused with those of the opposite leaf while the base of the stipules is also fused to the tissue of the stem to form a sheath around the node. The free parts of the stipules are apiculate.
The nodes are oval in transverse section ( Figure 17) and the general anatomy of the node agrees with that of the internode except that in the region of the leaf gap there is no crystal sclerenchyma.
The node is the same for all the species studied and is unilacunar with one trace (Figure 17). Each leaf trace forms two lateral (secondary) branches (Figure 18), each of which branches again. The outer (tertiary) bundles run perpendicular to the original leaf trace, into the stipule ( Figure 19) and fuse with the corresponding bundles from the opposite leaf trace in the apex of the stipules (Figure 20). The inner (secondary) bundles constitute the two lateral veins in the petiole while the primary leaf trace forms the main vein of the petiole. In transverse section the main leaf trace appears semilunar. S.-Afr. Tydskr. Plantk., 1986, 52(3)

Discussion
The vegetative shoot apex The presence of glandular hairs on the stipules covering the apical meristem has already been mentioned by several authors (Lubbock 1890;Potter 1891;Groom 1893;Lubbock 1895Lubbock & 1897Esau 1960;Uphof eta/. 1962;Lersten & Curtis 1974;Fahn 1979) and is considered to be a characteristic feature of the family Rubiaceae by Schumann (1891), Solereder (1908), Humm (1944), Metcalfe & Chalk (1950), Bremekamp (1957), Wunderlich (1971) and Van Hove & Kagoyre (1974). Lersten & Horner (1967), Horner & Lersten (1968) and Lersten (1974a) described the glandular trichomes of Psychotria species in detail and Krause (1909) and Van Hove (1972) described those of Gardenia and Neorosea species respectively. Von Faber described the glandular hairs of Pavetta species as early as 1912, as did Lersten (1974b) for a number of Pavetta species, five from southern Africa. He called these hairs dendroid colleters. Based on Lersten's definition, dendroid colleters were positively identified in all the southern African Pavetta species studied. As the dendroid colleters of the different taxa studied did not differ significantly from one another, this character could not be used in the delimitation of these taxa. Horner & Lersten (1968) and Fahn (1979) reported the absence of a cuticle on the dendroid colleters but in this study, using Sudan Black B, a very distinct cuticle was demonstrated. This observation corresponds with Krause's (1909), Von Faber's (1912 and Uphof et al.'s (1962) observations. The glandular trichomes in the shoot apices of plants are thought to secrete a mucilaginous or resinous substance (Lubbock 1890;Groom 1893;Lubbock 1895;Solereder 1908;Von Faber 1912;Metcalfe & Chalk 1950;Esau 1960;Lersten 1974a & b;Fahn 1979). In some of the species in the family Rubiaceae this mucilage contains bacteria (Von Faber 1912;Boodle 1923;Humm 1944;Centifanto & Silver 1964;Lersten & Horner 1967;Horner & Lersten 1968;Van Hove 1972;Lersten 1974a;Lersten & Horner 1976). The secretion of mucilage and the occurrence of bacteria in the mucilage was not studied during this investigation but Von Teichman &   and Von Teichman eta/. (1982) described the presence of bacteria in the developing ovaries within the floral buds of Pavetta gardeniifolia.
The mucilage, which contains bacteria and is secreted by the dendroid colleters, was observed in the substomatal cavities ( Figure 9). It is, therefore, proposed that the bacterial leaf nodules are initiated by the entry of the bacteria and the mucilage into the leaf primordia via the early developing stomata (Herman 1983). This theory is similar to those proposed for the other nodulating species in the family S. Afr. J. Bot., 1986, 52(3) . . .

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• Figures 11 -16 Morphology of the internode of Pavetta species. 11. Transverse section through the internode of P. gardeniijolia var. gardeniifolia to illustrate the general anatomy; A -epidermis; B -lacunar collenchyma; C -parenchymatous cortex; D -periderm; E -vascular tissue; F-pith . 12. Transverse section through the internode of P. inandensis to show the phellem (G). The lipid rich cell walls are obvious after staining with Sudan Black; H -cuticle. 13. Longitudinal section through the internode of P. zeyheri to show the phellem (J). 14. Transverse section through the internode of P. gardeniifolia var. gardeniifolia to illustrate the fibres (K) and crystal sclerenchyma (L); M -phloem; N -crystal sand (polarized light). 15. Longitudinal section through the internode of P. gracilijolia to show the cell wall thickenings (P) in the crystal sclerenchyma cells; R -primary phloem fibres. 16. Transverse section through the internode of P. schumanniana to illustrate the crystal sclerenchyma (S) in the pith.

The internode
The general anatomy of the native Pavetta species agrees with that of the family Rubiaceae as described by Solereder (1908) and Metcalfe & Chalk (1950). The anatomy of the internode of the individual taxa studied does not differ significantly and cannot, therefore, be used in the delimitation of taxa.
The consecutive periderms described for P. inandensis, known as a rhytidome, is considered by Solereder (1908) and Metcalfe & Chalk (1950) to be characteristic of Pavetta species. The stem of this specific specimen must have been slightly older than those of the other specimens studied. Although this characteristic does not contribute to the delimitation of the Pavetta species studied, it confirms the presence of the rhytidome in the southern African Pavetta species, as previously described for the genus (Solereder 1908;Metcalfe & Chalk 1950). Solereder (1908) and Metcalfe & Chalk (1950) reported on the presence of crystal id;oblasts or crystal sclerenchyma in the internode of representatives of the family Rubiaceae. They described this tissue as having the crystals embedded in the cell walls. As described in this paper, the cells develop thick walls and the crystals are contained in the cell lumen. Parameswaran & Schultze (1974) Horner (1980) the presence and shape of crystals can be of diagnostic value and the occurrence of two or more types of crystals in taxa might be of particular importance for classification. In Pavetta, where both crystal sand and rhomboid crystals are found in different tissues, these structures may be taxonomically significant. Here again these features do not contribute to the delimitation of the Pavetta species studied. However, these characters can be used at the genus, family and subfamily levels. Various authors (Franceschi & Horner 1980) reported the relationship between the presence of crystals and bacterial nodules and propose that the bacteria utilize the crystals. This hypothesis has not been examined during this study.
Although the individual characters of the internode cannot be used in the delimitation of taxa, in combination, these characters can be used for identification at the generic level. According to Metcalfe ( 1942-3) the position of the periderm and the pericyclic sclerenchyma in the stem can be of taxonomic value.

The node
The interpetiolar stipules of the Rubiaceae are considered to be the result of fusion of four independent stipules from the two opposite leaves (Sinnott & Bailey 1914;Bierhorst 1971;Neubauer 1981).
The unilacunar node of Pavetta species and most other representatives of the family Rubiaceae is considered to be a reduction of the presumably more primitive trilacunar node (Sinnott 1914;Sinnott & Bailey 1914) and is, therefore, phylogenetically more advanced. Most plants with trilacunar nodes possess stipules, whereas stipules are usually absent in plants with unilacunar nodes . About 20 of the families with unilacunar nodes have members with stipules but in most of these cases the stipules are reduced. According to Sinnott & Bailey (1914) it is to be expected that stipules should occasionally persist in families with unilacunar nodes derived from a trilacunar ancestor. This is further underlined by the fact that some genera in the family Rubiaceae have trilacunar nodes (Howard 1970) whereas some genera are unilacunar but with three leaf traces (Ridsdale 1978). Dahlgren (1975) showed a close relationship between the orders Oleales (Oleaceae) and Gentianales (containing among others the family Rubiaceae) under the superorder Gentiananae. This relationship is partly based on anatomical and gross morphological characters. Unilacunar nodes and exstipulate, entire leaves are found in the families Oleaceae and Oliniaceae whereas mainly unilacunar nodes and stipulate entire leaves are found in the family Rubiaceae (Sinnott & Bailey 1914). The unilacunar node and stipulate entire leaves of Pavetta species described in this paper serve as a further example of this relationship between the families Oleaceae and Rubiaceae. Another possible relationship is indicated between the families Rubiaceae and Oliniaceae (Herman 1983).
The pattern of vascular supply to the stipules described for Coffea arabica by Varossieau (1940in Howard 1970) is similar to that found in the Pavetta species reported on here. Bierhorst (1971) and Neubauer (1981) also described this type of vascular supply to interpetiolar stipules.
Although the nodal structure is the same for all the Pavetta species studied and therefore not important in the delimitation of taxa, it is important for intergeneric and interfamilial comparisons. It serves furthermore to explain the vascularization of the petiole which will be reported on in a subsequent paper.

Conclusion
The aim of this study was to examine the anatomy of the vegetative shoot apex and young stem of the southern African Pavetta species with a view to contributing to the taxonomic delimitation of the taxa. It became clear that none of the features studied could be used for identification purposes at the species level as the characters were constant in all the species studied.