Pollen morphology of members of southern African Boerhavia and Commicarpus (Nyctaginaceae)

In southern Africa, Boerhavia L. and Commicarpus Standl. are the most species-rich genera of the Nyctaginaceae. Hitherto, the pollen morphology of only three southern African species of each of these genera has been described. A palynological study of the remaining species is therefore necessary to complete the information for the taxa. The pollen morphology of sixteen species was studied with light, scanning electron and transmission electron microscopy. Pollen grains of Boerhavia and Commicarpus are uniform in shape and sculpturing. Grains are spheroidal and pantoporate, and the tectum tubuliferous and spinulose. Pollen grains of the Boerhavia are 52–91 μm in diameter and those of Commicarpus 52–129 μm. Spinules in both genera are 1–5 μm long. Pore plates have one or two spinules. In both genera the exine is 3.5–11.5 μm thick, the tectum 0.9–3.9 μm thick and the collumellae 0.6–2.8 μm long. Foot layers are 0.9–5.1 μm thick and the endexine is barely visible. Size variation of the pollen grain, pore diameter and exine thickness overlap and cannot be used to distinguish between the two genera or the individual species.


INTRODUCTION
Nyctaginaceae Jussieu, commonly known as the fouro'clocks, are a relatively small family of about 30 genera and 300-400 species (Levin et al. 2001;Douglas & Manos 2007).It is distributed throughout the tropical and subtropical regions of the New World (Bittrich & Kühn 1993;Jordaan 2000), but mainly in the Americas (Stannard 1988).In southern Africa, south of the Zambezi River (Botswana, Lesotho, southern Mozambique, Namibia, South Africa, Swaziland and Zimbabwe), fi ve genera and 20 species occur of which sixteen species are found in Namibia (Germishuizen & Meyer 2003;Struwig 2012).Namibia is therefore the centre of diversity for the family in southern Africa.Boerhavia L. and Commicarpus Standl.are the largest genera of the family in southern Africa, with seven and nine species respectively (Germishuizen & Meyer 2003).
Pollen grains of Nyctaginaceae are 17-200 μm in diameter, spheroidal, prolate or oblate and 3(4)-colpate, pantocolpate or pantoporate (Bittrich & Kühn 1993).Pores are covered by a pore plate, which can either be roughened or spinulose (Bittrich & Kühn 1993).The exine is very thick and varies in sculpturing (spinulose, tubuliferous or coarsely reticulate) (Nowicke 1970;Bogle 1974).Heimerl (1934) used pollen morphology to divide the Nyctagineae into four subtribes.Subtribe Nyctagininae (to which Boerhavia and Commicarpus belong) is characterised by large, spheroidal, pantoporate pollen grains with thick walls and a tubuliferous and spinulose exine (Nowicke 1970;Nowicke & Luikart 1971).Nowicke (1970) found that the shape and size of the grains, and the thickness of the exine, form a continuum within gen-era and species of subtribe Nyctagininae, so much so that these characters are of limited taxonomic significance.Nowicke & Luikart (1971) came to the same conclusion for the other subtribes.Recently, a phylogenetic study of the family (Douglas & Manos 2007) found that pollen morphology is homoplasious among genera and can therefore not be used to support the tribal and subtribal divisions of Heimerl (1934).
Pollen grains of 13 of the 40 species of Boerhavia and three of the 35 species of Commicarpus were described by Nowicke (1970) as spheroidal, pantoporate and the sexine as tubuliferous and spinulose.The spinules are 1.0-2.5 μm long.Grains of Boerhavia are 70-138 μm in diameter, with 18-40 pores.Pores are 2.4-7.0 μm in diameter and the pore plates have a roughened base with 1 or 2 spinules.Sexine is 2.5-6.0 μm thick and the nexine is 4-7 μm.Grains of Commicarpus are 84-112 μm in diameter with 27-39 pores.Pores are 3.0-5.5 μm in diameter and the pore plates have 1 or 2 spinules.Sexine is 2.5-5.5 μm thick and the nexine is 2.5-5.5 μm.(Nowicke 1970;Perveen & Qaiser 2001).A palynological study was therefore conducted to provide a detailed description of the pollen morphology of other native members.As previous studies (Nowicke 1970;Perveen & Qaiser 2001) have suggested that pollen morphology of these two genera is uniform and of limited taxonomic value at the generic or species level, the poorly known southern Africa species provided an opportunity to report further evidence regarding the taxonomic signifi cance of palynological characters.

MATERIALS AND METHODS
Pollen from herbarium specimens and fresh plant material collected in situ in Namibia and South Africa Bothalia 43,1 (2013) was investigated with light microscopy (LM), scanning electron microscopy (SEM) and transmission electron microscopy (TEM).
Pollen grains were acetolysed according to the method of Erdtman (1969).For LM, pollen was mounted in glycerine jelly and sealed with entellan (Product 7961, E. Merck, Darmstadt) according to the method of Fripp (1983).For SEM, a drop of 96% ethanol/pollen mixture was pipetted on specimen stubs, dried and sputter-coated with gold/palladium.Specimens were examined with a FEI Quanta 200 Environmental Scanning Electron Microscope (ESEM) or a JEOL JSM 840 SEM.A minimum of eight pollen grains from three individuals per species were used to measure the diameter of the grain, pores and the length of the spinules.
For TEM, unacetolized grains were fi xed in 4% aqueous paraformaldehyde and rinsed in three changes of 0.05 M cacodylate buffer, pre-stained in 2% uranyl acetate (pH 2), dehydrated in a graded ethanol series and embedded in resin (L.R. White™ Wirsam/London Resin Company).Sections were made with a Reichert-Jung Ultracut E microtome and contrasted with 2% uranyl acetate (pH 2) and lead citrate (Anala R).Sections were examined with a Philips CM10 Transmission Electron Microscope.

DISCUSSION
Pollen grain size range in the southern African species of Boerhavia is smaller than but overlapping the range reported by Nowicke (1970) for Boerhavia from the Americas (Table 1; Table 2).Nowicke (1970)  Africa, and recorded the diameter of the grains to be nearly twice as large as that measured for the present study (Table 1; Table 2).The shape and sculpturing are, however, the same.Nowicke (1970) also studied three Commicarpus species, namely C. brandegei from Mexico, and C. fruticosus and C. pentandrus from southern Africa.The pollen shape and sculpturing of the latter two (Nowicke 1970) correspond with the fi ndings of the present study, although the size range measurements of the grain diameters are less than that measured by Nowicke (1970) and the pore diameter is larger than that measured by Nowicke (1970) (Table 1; Table 2).Perveen & Qaiser (2001) studied the pollen morphology of the Nyctaginaceae in Pakistan and included species also found in southern Africa, namely B. diffusa var.diffusa, B. repens subsp.repens and C. helenae var.helenae.The present study confi rms the pollen shape and sculpturing of these three species given by Perveen & Qaiser (2001), but in some instances the diameter of the pollen grains, the pore diameter and the exine thickness differ.Slight differences in measurements between this study and that of Perveen & Qaiser (2001) can be ascribed to natural variation within the species and between geographical areas.
Pollen morphology (shape and sculpturing) of Boerhavia and Commicarpus is not diagnostic, and although pollen grains of the Boerhavia species are smaller than those of the Commicarpus species (Table 1), the ranges overlap substantially, making it impossible to distinguish between the genera with the aid of numerical measurements alone.Pore diameter of Commicarpus species has a higher range than that of Boerhavia species and the exine of Commicarpus species has a lower range in thickness than that of Boerhavia species (Table 1).However, the overlap in size ranges of the pollen grains, pore diameter and thickness of the exine does not provide suffi cient discontinuity to distinguish between the two genera, nor the individual species.

CONCLUSION
The pollen morphology of ten southern African species of Boerhavia and Commicarpus was described for the fi rst time and proved to be essentially uniform in shape and sculpturing.Pollen grains are spheroidal and pantoporate and the tectum is tubuliferous and spinulose.Pollen grains of Boerhavia are generally smaller than those of Commicarpus species.Pore diameter in Commicarpus is generally larger than that in Boerhavia and the exine in Commicarpus is thinner than in Boerhavia.However, the use of grain dimensions to distinguish among taxa is unreliable due to the extensive overlap in size ranges.
African members of Boerhavia and Commicarpus has not been described extensively, except for the naturalised B. diffusa var.diffusa and B. erecta, and the native B. repens subsp.repens, C. fruticosus, C. helenae var.helenae and C. pentandrus (Table 2).Pore diameters of B. diffusa var.diffusa and of B. repens subsp.repens (Table 1) show a slight overlap with the measurements of Perveen & Qaiser (2001) (Table 2).Exine of B. diffusa var.diffusa (Table

Table 1 .
-Measurements of pollen grains of