Pollen morphology of Petopentia and Tacazzea (Periplocaceae)

The pollen morphology of five species of Tacazzea Decne. and the monotypic genus Petopentia Bullock has been studied . All the species of the two genera are characterized by pollen grains arranged in tetrads. The arrangement of the grains may be rhomboidal, tetragonal, linear, T-shaped, tetrahedral or decussate. Linear and T-shaped tetrads were only observed in Petopentia. The 4-6 pores are restricted to the junction area of adjacent grains. Exine is smooth and covered with a thin electron-dense layer. Endexine is present above the well-developed intine. The internal walls are perforated. The pollen grains of tetrads are connected by wall bridges (cross-wall cohesion). Petopentia and Tacazzea can be distinguished on pollen morphological characteristics.


Introduction
The family Periplocaceae was formerly a subfamily (Periplocoideae) of the Asclepiadaceae, but raised to family status by Schlechter (1924), Bullock (1957), Hutchinson (1959) and Dyer (1975) . Of the 50 genera included in the Periplocaceae, 18 occur in Africa. Among these, Raphionacme Harv. is the largest genus with 35 species endemic to Africa (Verhoeven & Venter 1988). Raphionacme is also the only herbaceous genus, although a number of its species are climbers. Most of the other genera are lianous and a small number are shrubs.
The African taxa of the Periplocaceae are under taxonomic revision by the present authors . This paper on the pollen of Petopentia and Tacazzea constitutes part of a comprehensive palynological investigation of the African taxa of the Periplocaceae to determine whether morphology of the pollen could supplement taxonomic evidence in a systematic study of the genera, and to present results which might be considered in a future clarification of the affinities of the genera in the family. The use of a wider range of characters frequently results in a clearer and more accurate understanding of generic limits.
In their study on the pollinaria of the Asclepiadaceae Schill & Jakel (1978) investigated 408 species in 114 genera by light, scanning and transmission electron microscopy, and showed that the morphology of the pollinaria of the Periplocoideae differs from that of the Cynanchoideae. The Periplocoideae have pollen grains united in tetrads, whilst the Cynanchoideae have pollinia consisting of single pollen grains. The translators of the Periplocoideae are spoon-shaped or cornet-shaped with a viscidium attached. The pollinaria of the Cynanchoideae consist of a corpuscle and two caudicles. Schill & Jakel (1978) investigated nine genera of the Periplocoideae. Data on tetrad size and number of pores for T. venosa and P. natalensis are given by Schill & Jakel (1978).

Taxonomic aspects and distribution of Petopentia and Tacazzea
Petopentia is a monotypic genus. P. natalensis (Schltr.) Bullock is found in subtropical Natal (South Africa) only. It is a liane associated with rocky habitat in places of moderate precipitation and climate. The flowers are much larger than those of Tacazzea and are yellowishgreen in colour.
Tacazzea comprises five species (Table 1). T. apiculata Oliv. is a large liane of tropical moist forests and is most widely distributed all over tropical and subtropical Africa wherever swampy conditions occur. Its ability to maintain itself in a variety of situations is reflected in its great and often confusing morphological variability. T. conferta N.E. Br. is also a large liane found on the fringes of mountain forests, especially the bamboo forests at 2 000-3 000 m above sea level. Both T. apiculata and T. conferta bear conspicuous sprays of reddish to greenish-yellow multi-flowered cymose inflorescences. T. tomentosa Bruce is rather rare and inhabits savanna in Uganda, Sudan and Ethiopia. It is a climber over low trees and bears small cymes of creamy to T. venosa is found in Ethiopia and T. rosmarinifolia in Angola. Both occur in riverbank vegetation and have large multi-flowered inflorescences with red-yellow flowers.

Materials and Methods
grains (embedded in agar after osmium tetroxide fixation) were fixed in 3% phosphate-buffered glutaraldehyde (0,1 mol dm· 3 phosphate buffer, pH 7,0), postfixed in 1 % osmium tetroxide , dehydrated in ethyl alcohol and embedded in Spurr's low viscosity resin. Sections were cut with a diamond knife , stained with uranyl acetate, followed by lead citrate , and examined with a Philips 300 electron microscope at 60 kV. Pollen was taken from either herbarium specimens or from material collected in natural habitats. Pollen sources are indicated in Table 1. For both light microscopy (LM) and scanning electron microscopy (SEM) study, pollen was acetolysed according to the method of Erdtman (1960) and prepared and investigated as described by Verhoeven & Venter (1988) . Data on the measurements of length and width of rhomboidal tetrads were subjected to analysis of variance and significant differences were identified with the aid of Tukey's test (Steel & Torrie 1980) .
For transmission electron microscopy (TEM) fresh material was used. Only T. apiculata and P. natalensis were examined by TEM . Pollen carriers and pollen

Pollen morphological descriptions
The terminology of Faegri & Iversen (1964) was used in the description of the pollen grains .

Petopentia
Pollen grains are united in uniplanar tetrads arranged either rhomboidally (Figures 1 & 5) , linearly (Figures 2 & 6) , or T-shaped (Figures 3 & 7) . A great number of pollen grains are also arranged with three grains in a row and the fourth one slightly underneath (Figures 4 & 8) . It looks like a transition form between linear and T-shaped on one hand and rhomboidal on the other. The size of  Table 2). Individual grains of the tetrads have 4-6 pores, although the middle grains of the linear and T-shaped tetrads may have 8 pores. The pores are round, semi-circular or irregular and are usually opposite to each other and restricted to the junction area of adjacent grains ( Figure  1). Single pores not associated with the junction area of adjacent grains are often found at the distal end of terminal grains of tetrads (Figure 4), or in middle grains of linear and T-shaped tetrads (Figures 2 & 3). Pores are sometimes covered with a thin layer of exine material (Figure 1).

Exine structure of Petopentia and Tacazzea
The exine structure is the same in both genera. The exine is smooth and covered with a thin electron-dense layer ( Figure 18). Ektexine and endexine have the same electron density but end exine has an irregular appearance because of channels which occur throughout the endexine ( Figure 19). In Petopentia the ektexine is slightly thicker than the endexine but in Tacazzea the endexine is very thin in comparison to the ektexine (Figures 18 & 19). The intine is well developed with often a two-layered structure because of the loose fibrillar structure of the outer layer ( Figure 18). The internal walls are perforated ( Figure 17) and have the same structure as the exterior wall. The common wall between adjacent grains has wall bridges comprising intine and endexine (Figures 20 & 21). Starch grains are absent from the cytoplasm.

Discussion
The pollen morphology of the family Periplocaceae has rarely been investigated. Verhoeven & Venter (1988) reported on the pollen morphology of 35 Raphionacme species while Lebrun et al. (1984)    Chev. (Asclepiadaceae) as R. bingeri (Periplocaceae) was based on the presence of tetrads and absence of pollinia. In their study on the pollinaria of the Asclepiadaceae Schill & Jakel (1978) investigated the following representatives of the Periplocaceae: Tacazzea, Periploca L., Parquetina Baill., Cryptostegia R. Br., Hemidesmus R. Br., Ectadiopsis Benth., Raphionacme, Zygostelma Benth. and Omphalogonus Baill. Data on tetrad size (seven genera) and number of pores (four genera) are given. Compound pollen grains occur in more than 56 families of angiosperms (Erdtman 1945;Walker & Doyle 1975;Knox & McConchie 1986). The cohesion mechanisms in mature polyads were discussed by Knox & McConchie (1986). The cohesion of compound pollen occurs by attachment of the tectum (simple cohesion) or by connecting wall bridges (cross-wall cohesion). Crosswall connection is known to account for cohesion in at least ten families (Periplocaceae not included) (Knox & McConchie 1986). In this cohesion mechanism wall bridges are present in the common wall between adjacent grains, and these bridges may comprise several wall layers. In Petopentia and Tacazzea the bridges comprise intine and endexine. The same wall layers were also observed in Raphionacme (Verhoeven & Venter 1988). The family Periplocaceae can thus be added to the ten families where cohesion occurs by wall bridges. The intine wall bridges also indicate the position of pores in the acetolysed pollen grains. Guinet (1965) observed pores in the internal walls of representatives of the Burmanniaceae, Caesalpiniaceae, Hydrostachyaceae and Asclepiadaceae, and interpreted the pores as a tendency towards the disappearance of these walls. Although this type of evolutionary development, from a tetrad to a monad, is an idea which is in conflict with the usual view, in some instances monads may have secondarily evolved in this way from tetrads and in such cases solitary grains represent an advanced rather than a primitive character-state (Walker & Doyle 1975).
Pollen tetrads and polyads are common in a number of families and-have been used in systematic treatments to separate genera and species (Oldfield 1959;Skvarla et al. 1975;Takahashi 1986). In the Mimosoideae extensive use of tetrads and polyads is made to separate genera (Guinet 1981a, b;Niezgoda et al. 1983). In the Periplocaceae Raphionacme can be distinguished from Petopentia and Tacazzea by the 10-16 pores per pollen grain as against 4--6 in Petopentia and Tacazzea . Four to six pores were also recorded by Schill & Jakel (1978) for Parquetina and Periploca. The mono typic Petopentia natalensis can be distinguished from Tacazzea by the presence of linear and T-shaped tetrad arrangements which were not observed in Tacazzea. The average rhomboidal tetrad size of 71 x 36 f-lm also distinguishes Petopentia from Tacazzea where average rhomboidal tetrad size varies from 39-63 x 25-44 f-lm in the different species. In Tacazzea, tetrad arrangement is very similar in T. apiculata, T. rosmarinifolia and T. venosa. The majority of grains are arranged tetrahedrally or rhomboidally with the rhomboidal tetrads rectangular to spherical in form. T. venosa differs significantly from T. rosmarinifolia and T. apiculata in the length of rhomboidal tetrads. The arrangement of tetrads is mainly rhomboidal in T. conferta and T. tomentosa. T. tomentosa differs significantly from all the other taxa by the width of the rhomboidal tetrads and from T. conferta by the length of the rhomboidal tetrads. Petopentia and Tacazzea are characterized by starchless pollen grains, as is the case with all the Periplocaceae. According to Baker & Baker (1979) starchy pollen is a feature of This study has revealed that Petopentia, Tacazzea and Raphionacme can be distinguished on pollen morphological characteristics: Raphionacme by the 10-16 pores as against 4-6 in Petopentia and Tacazzea, Petopentia by the presence of linear and T-shaped tetrad arrangement, and Tacazzea by the 4-6 pores per pollen grain and absence of linear and T-shaped tetrad arrangement. Studies on the other genera, which are in progress, will however show whether all the African genera of the Periplocaceae can be distinguished from each other on pollen morphological characteristics. It is hoped that these pollen data will add to the systematic information on this family and will be used for comparisons within the family.