Pollen morphology of Erodium in southern Africa

S. Afr.. Bot., 987,5(): 79-8 79 Pollen morphology of Erodium in southern Africa R.L. Verhoeven* and H.J.T. Venter Department of Botany, University of the Orange Free State, P.O. Box 9, Bloemfontein, 900 Republic of South Africa Accepted 5 March 987 The pollen morphology of seven of the eight species of Erodium L'Herit. (Geraniaceae), that are found in southern Africa, has been studied. The pollen grains are tricolporate, prolate spheroidal to suboblate. The exine structure is reticulate to striate. Only the Erodium type of pollen was observed. The nexine consists only of nexine (footlayer). Die stuifmeelmorfologie van sewe van die agt spesies van Erodium L'Herit. (Geraniaceae) wat in suidelike Afrika aangetref word, is bestudeer. Die stuifmeelkorrels is trikolporaat, prolaat-sfero'idaal tot suboblaat. Die eksien besit 'n geretikuleerde tot gestrieerde struktuur. Slegs die Erodium-tipe van stuifmeel is waargeneem. Die neksien bestaan slegs uit neksien (voetlaag). Keywords: Erodium, Geraniaceae, pollen morphology 'To whom correspondence should be addressed Introduction The family Geraniaceae includes the five genera, Erodium L'Herit., Geranium L., Monsonia L., Sarcocaulon (DC.) Sweet and Pelargonium L'Herit. (Hutchinson 969). The family is divided into two tribes, the Geranieae, which includes Erodium, Geranium, Monsonia and Sarcocaulon, the flowers of which are all basically actinomorphic, while Pelargonium, with zygomorphic flowers, is the sole genus of the Pelargonieae. The common diagnostic feature of all five genera is the schizocarpic fruit. Erodium is distinguished from the Table other genera by the five fertile stamens and five staminodes. Erodium includes about 60 species of annual or perennial herbaceous plants. Leaves are pinnate or undivided to pinnatifid to pinnatisect. The size and shape of leaves can vary considerably between populations and variation is often also pronounced within populations (Dahlgren 980). The centre of distribution is the Mediterranean region where 6 species occur, with an extension into Central Europe. Only seven species are not found in either Europe or the Mediterranean region. Of these one is in Central Asia, one in East Asia, one Species and voucher specimens from which pollen was studied Collector Locality Herbarium botrys (Cav.) Berto!. Botha & Coetzee 679, Oct. 976 C. Boucher 90, 0 Sept. 978 J. Marsh 70, 0 Sept. 968 T. Salter 96, Oct. 95 Hopefield (8 AB) Stellenbosch (8 DD) Stelenbosch (8 DD) Wynberg (8 AB) PRU SAM chium (Burm. f.) Willd. T. Salter 707, Oct. 97 J. Hutchinson 77, 6 Sept. 98 R. Parker BH 7, 9 Aug. 98 C. Smith 07, Nov. 96 Hout Bay (8 AB) Saldanha Bay (7 BB) Kogel Bay (9 BD) Nacht Wacht (0 CA) cicutarium (L.) L'Herit. M. Steyn 68, Oct. 99 R. Verhoeven 6, 8 July 98 P. Range 86, Sept. 9 J. Prosser 90, 5 Aug. 9 A. Leeuwenberg, 5 Aug. 98 A. Leeuwenberg 585, 6 June 98 Kirstenbosch (8 CD) Paarl (8 DB) Kuibis (66 DB) Salford, UK Wageningen, Netherlands La Cabanasse, France NBG SAM UPE incarnatum (L.) L'Herit. Oliver 58, Nov. 97 F. Leighton 806, 8 Nov. 9 C. Boucher 000, 8 Nov. 97 L. Willems 78, 0 Nov. 958 Villiersdorp (9 CD) Arieskraal (9 BD) Kogelberg (9 BD) Houwhoek (9 AA) NBG malacoides (L.) L 'Herit. R. Verhoeven 9, Dec. 98 R. Verhoeven, 5 Dec. 98 J. v.d. Walt 88, Oct. 977 Bellville (8 DC) Bellville (8 DC) Caesars Dam (5 DA) -U moschatum (L.) L'Herit. J. v.d. Walt 7, 9 Sept. 975 J. v.d. Walt 79, Aug. 977 R. Verhoeven 6, 8 July 98 H. Taylor 78, Aug. 96 H. Fourcade 8, Oct. 9 Blackheath (8 DC) Piekenierskloof (8 DB) Paarl (8 DB) Swellendam (0 AB) Wittedrif ( AB) -U -U venteri Guittonneau & Verhoeven J. v.d. Walt 895, Oct. 977 D. Johnstone, Oct. 99 Hilliard & Burtt 660, 9 Feb. 97 Cradock (5 BA) Hattingspruit (80 AA) Naudesnek (08 CA) -U NU ruthenicum M. Bieb. No pollen available

S.-Afr. Tydskr. Plantk., 987, 5() 80 in South Africa, two in western North America, one in South America and one in Australia (Warburg 98). In southern Africa eight species have been identified of which only incarnatum (L.) L'Herit. and venteri Guittonneau & Verhoeven are indigenous. At species level the taxonomy of flora Europaea (Tutin et al. 968) was followed. In a preliminary study Bortenschlager (967) showed that the pollen morphology of the Geraniaceae varies. Since the family is under revision [Pelargonium (Van der Walt 977, 98; Van der Walt & Vorster 98; Vorster 986; Vorster & Van der Walt 98), Monsonia already completed by Venter (979) and Sarcocaulon by Moffett (979)], this investigation was undertaken to determine whether pollen morphology could supplement taxonomic evidence from a systematic study of the southern African Erodium species. Materials and Methods For this study, pollen was taken from either herbarium specimens or from material collected in natural habitats. The collectors and the localities where materials were obtained are shown in Table. Seven southern African species were studied (Table ). Pollen of ruthenicum M. Bieb. was not available. For both light microscopy (LM) and scanning electron microscopy (SEM) study, pollen was acetolysed according to the method of Erdtman (960). Acetolysed samples for SEM were rinsed in acetic acid, washed twice in water, air-dried on stubs, coated with gold and examined with an lsi 00 SEM at 0 kv. For light microscopy the remainder of acetolysed material was prepared according to the method of Reitsma (969). Samples were examined with a Zeiss Photomicroscope. Table Results Pollen grains are tricolporate, prolate spheroidal to suboblate. In size they range from 6 to 85,5!lm (polar diameter) and,5 to 79,5!lm (equatorial diameter) (Table ). Ora are round to lolongate. The ora are generally well delimited towards the poles but less distinctly so laterally (Figure ). Exine structure is semitectate and reticulate to striate (Figures - 5). Supratectal processes are absent. A striate pattern where the lirae are interwoven (Figure 5), and width of lirae and striae the same, is the most common exine pattern among the species investigated. In the same species however a range of exine variation from striate interwoven to reticulate (Figures 6 & 7) was observed. The striate interwoven to reticulate exine structure was observed in botrys, (Cav.) Bertol., chium (Burm. f.) Willd., cicutarium (L.) L'Herit., malacoides (L.) L'Herit. and moschatum (L.) L'Herit. In incarnatum and venteri the striate pattern Diameter of pollen grains (!lm) 78,8 (75-8) 76,7 (75-8) 7,6 (70,5-79,5) 78,9 (75-85,5),,6,9 7,6 (69-79,5) 69, (6-75) 7, (67,5-76,5) 76, (69-8),8,7,9,7 5 6 59,9 q(57-6) (60-67,5) 6 58,6 (57-60) 59 (55,5-6) 6,7 (60-6) 6, (60-67,5),,,7,,5 65, (60-70,5) 68 (60-7,5) 65 (6,5-67,5) 6, (60-70,5),8 65,6 65,5 65,7 5 59, (57 (57 59 59,8 (57 58, (5,5 6,6 (57-0,9 (7,5-5) 7, (6-9) 7,5 (6-) chium circutarium incarnatum Mean for specimens Polar diameter botrys malacoides moschatum venteri Measurements of pollen grain size are based on a minimum of 5 grains per specimen. A complete set of slides is filed in the Department of Botany, University of the Orange Free State, Bloemfontein. For transmission electron microscopy (TEM) fresh material was used. Anthers were fixed in 070 phosphate buffered glutaraldehyde (0, mol dm phosphate buffer, ph 7,0), postfixed in 070 phosphate buffered osmium tetroxide, dehydrated in ethyl alcohol, followed by two changes of propylene oxide and embedded in Spurr's low viscosity resin (Spurr 969). Sections were made with a diamond knife, stained with uranyl acetate and then lead citrate (Reynolds 96). (60-7),8 (6-70,5), (6-69),8 6) 6,5) 6) 60) 67,5) 76,8, 7,6,6 60,9,6 66,, Equatorial diameter 78,5 (78-79,5) 76 (7,5-79,5) 7,9 (70,5-75) 78,5 (78-79,5) 0,9,,9 0,9 7, (69-79,5) 69, (66-75) 7 (67,5-75) (69-75) 7,5,, 60,6 6, 59, 59,6 6,9 6,5 (58,5-6,5) (6,5-67,5) (58,5-60) (58,5-6,5) (60-66) (6,5-66),,9 0,8,, 67,5 (6-7) 69, (6,5-7) 6,8 (60-67,5) 65,5 (6,5-67,5),5,5,9,7 65,6, 65,8 (6,5-69),9 66, (6,5-69) 65, (6-67,5),9,,8,,, Mean for specimens 75,6, 7,5 6,8, 67,, 65,8, 59,9, 59,5 (57-6,5) 58,6 (57-60) 60,5 (58,5-6) 58, (57-60) 6,5 (58,5-6),,,, 59,7,8 8,8,6, (9-6,5) 7,6 (,5-0,5),8 0 (6 -,5), 9,9,8

8 S. Afr. J. Bot., 987, 5() Figures - 5 Scanning electron micrographs of Erodium species.. incarnatum (Boucher 000), equatorial view.. moschatum (Van der Walt 7), polar view.. moschatum (Van der Walt 7), detail of reticulate exine.. cicutarium (Steyn 68), polar view. 5. cicutarium (Steyn 68), detail of striate interwoven exine. Scale in polar view equals 0 m and m in detail figures. is formed by parallellirae which are often dividing. Adjacent lirae are interconnected by thinner lirae (Figures 8 & 9). The exine is divided into sexine and nexine, which are of similar electron density. Sexine layering is as usual, with sexine, to 5,9!lm thick (Table ), attached to the nexine (0,6 to,!lm thick) by means of columellae which unite into a tectum above (Figure 0). Sexine, nexine and intine thicknesses were taken from thin sections. Nexine consists only of nexine I (footlayer) with traversing channels present (Figure 0). The intine is 0,6 to,8!lm thick, and starch grains are present in the cytoplasm. Discussion Bortenschlager (967) examined Erodium species and recognized two basic types, namely Geranium multiflorum and Erodium. The Geranium multiflorum type is characterized by supratectal processes and was observed by Bortenschlager (967) in bryoniaefolium Boiss., guttatum (Desf.) Willd., and texanum Gray. In the Erodium type three subtypes, Corsicum, Gruinum and Hirtum, were identified on the basis of exine sculpture. Characteristic of the Corsicum subtype is the reticulate exine, Gruinum SUbtype the striate exine with dividing lirae and characteristic of the

S.-Afr. Tydskr. Plantk., 987, 5() 8 Figures 6-9 Scanning electron micrographs of Erodium species. 6. malacoides (Van der Walt 88), detail of striate interwoven exine. 7. malacoides (Verhoeven 9), detail of reticulate exine. 8. incarnatum (Boucher 000), polar view. 9. incarnatum (Boucher 000), detail of parallel and dividing lirae. Scale in polar view equals JO ).m and I ).m in detail figures. Table Exine and intine thickness (!lm) botrys cicutarium malacoides moschatum Sexine Nexine Intine, - 5,,7-5,6, - 5, 5, - 5,9 0,6-0,9 0,6-0,9 0,6 -, 0,9 -,, -,8 0,6-, 0,9-,8 0,6 Hirtum subtype the striate interwoven exine structure. In his examination of 5 Middle Eastern Erodium species of which of Bortenschlager's species were re-studied, EIOqlah (98) also identified the two basic pollen types, namely Geranium multiflorum and Erodium. The Geranium multijlorum type was observed in five species, namely bryoniaefolium, guttatum, arborescens (Desf.) Willd., litvinovi Woron., and oxyrchynchum M. Bieb. EI-Oqlah (98) demonstrated that a continuous range of sculptural variation occurs within the Erodium type of pollen which makes it impossible to identify subtypes. In grassifolium L'Herit. considerable sculptural variability was recorded to the extent that the E. hirtum SUbtype and gruinum subtype were recorded in different samples of the same species. It is thus concluded by EI-Oqlah (98) that it is impossible to recognize discrete subtypes simply on tectal sculpture. In this study sculptural variation was observed in the same species between striate interwoven exine (Hirtum subtype) and T 0 Figure 0 Transmission electron micrograph of mesocolpial view of cicutarium (Verhoeven 6) pollen wall. C - columella; I - intine; N - nexine; T - tectum. Bar = ).m. reticulate exine (Corsicum subtype), but not between the Hirtum and Gruinum subtype as observed by EI-Oqlah (98). This study revealed that subtypes as found by BortenschIager (967) can be distinguished, but because of a range of

S. Afr.. Bot., 987,5() sculptural variation between subtypes in the same species, subtypes cannot be used for identification and intermediate forms are difficult to place in a specific subtype. Acknowledgements The financial support by the Council for Scientific and Industrial Research, Pretoria, and the University of the Orange Free State is gratefully acknowledged. References BORTENSCHLAGER, S. 967. Vorliiufige Mitteilungen zur Pollenmorphologie in der Geraniaceen und ihre systematische Bedeutung. Grana palynol. 7: 00-68. DAHLGREN, G. 980. Cytological and morphological investigation of the genus Erodium L.'Her. in the Aegean. Bot. Notiser : 9-5. EL-OQLAH, A.A. 98. Pollen morphology of the genus Erodium L'Herit. in the Middle East. Pollen et Spores 5: 8-9. ERDTMAN, G. 960. The acetolysis method. A revised description. Svensk. bot. Tidskr. 5: 56-56. HUTCHINSON, J. 969. Evolution and phylogeny of flowering plants. Academic Press, London. MOFFETT, R.O. 979. The genus Sarcocaulon. Bothalia : 58-6. REITSMA, T. 969. Size modification of recent pollen grains under different treatments. Rev. Palaeobot. Palynol. 9: 75-0. 8 REYNOLDS, S. 96. The use of lead citrate at high ph as an electron-opaque stain in electron microscopy.. Cell. Bioi. 7: 08-. SPURR, A.R. 969. A low-viscosity epoxy resin embedding medium for electron microscopy.. Ultrastruct. Res. 6: -. TUTlN, T.G., HEYWOOD, V.H., BURGESS, N.A., MOORE, D.M., VALENTINE, D.H., WALTERS, S.M. & WEBB, D.A. 968. Flora Europaea, Vol., pp. 99-0, Cambridge University Press, Cambridge. VAN DER WALT, J.J.A. 977. Peargoniums of southern Africa, Vol., Purnell, Cape Town. VAN DER WALT, J.J.A. 98. A new species of Pelargonium (Geraniaceae) from the south-western Cape. S. Afr.. Bot. : 56-58. VAN DER WALT, J.J.A. & VORR, P.J. 98. Pelargoniums of southern Africa, Vol., Juta, Cape Town. VENTER, H.J.T. 979. A monograph of Monsonia L. (Geraniaceae). Meded. Landbouhoogesch. Wageningen, Nederland 79(9): - 8. VORR, P. 986. Pelargonium desertorum (Geraniaceae): a new species from the north-western Cape Province. S. Afr.. Bot. 5: 8-86. VORR, P. & VAN DER WALT, J.J.A. 98. Two new species of Pelargonium L'Herit. (Geraniaceae) from South Africa. S. Afr. J. Bot. : 76-8. WARBURG, F. 98. omy and relationship in the Geraniales in the light of their cytology. New Phytol. 7: 89-0.