246 S. AIr. j, Bot. 1998,64(4): 24&-249

The leaf anatomy of two Clerodendrum species (Verbenaceae)

P,P,J , Herman National Botanical Institute, Private Bag X101, Pretoria, 0001 Republic of South Africa e ~ mail: [email protected]

Received 16 March 1998: revised 9 JUlie 1998

The leaf anatomy of Clerodendrum triphyllum (Harv.) H.Pearson and C. louwalberfsii P.P.J.Herman is described. The leaves are amphistomatic, with mostry diacytic but also an isocytic, anomocytic and a few paracytic stomata. In cross section the leaves of C. louwalberlsii are dorsiventral but in C. triphyllum the mesophyll is homogeneous. The leaf surface of C. louwalbertsii is smooth, bulgy and undulate, whereas the surface of C. triphyflum is reticulate and less Du lgy. Peltate ha irs are scattered over both leaf surfaces of both species. The hairs have an 8-celled head, a unicellular stalk and a base cell wh ich is sunken below the level of the epiderm is.

Keywords: Anatomy . Clerodendrum, homogeneous, leaf surface.

Introduction (Figure IA and B), The stomata occur in the same plane as the Clerodendrum L. is probably th e largest genus in the family Ver­ rest of the epidermal cell s (Figure l A, Band 0) and are mostly benaceae (Moldenke 1973), with approximately 560 species and diacytic (Figure I C) but anisocytic, anomocytic and a few para­ varieties distributed mostly in A frica and Asia. Only about 20 cytie stomata were also observed. Peltate hairs occur in both the species arc native to the New World and many are cu ltivated as adaxial and abaxial epidermis. They consi st of a basal cell, ornamentals (Rueda 1993), The genus is represented by about 21 sunken below the leve l of the epidermis, a uni cellular stalk and species in southern Afri ca (Herman 1993). vary ing from trees 8-celled head (Figures 10, E and 20). In epidermal preparations, and shrubs to perennial herbs. epidermal cells radi ating around the base of the peltate hairs During preparation for a previous work (Retief & Herman were observed, more pronounced in C triphy/lum. In surface 1997), the material of inter alia Clerodendrum lriphyllum view the epidermal cells above and below the main and first (Harv.) H.Pearson housed in th e National Herbarium of the order veins are rectangular and arranged in parallel rows. This National Botanical Institute, Pretoria (PRE) was studied, and it arrangement is also characteristic of the epidermal cells along the became clear that there was more than one taxon represented in leafmargin, but the cells are more or less isodiametrical and reg­ the collection. C. IOlilValbertsii P.P.J.Herman was subsequentl y ular. The intercostal epidermal cells are irregular, vary in shape described as a new species (Herman 1995 ). The leaf morphol­ with straight cell walls (Figure I C), The cuticle of C. triphyllllm ogy, as reported here, was studied as additional support for the seems thicker th an that of C IOlfwalbertsii. recogniti on of two taxa. Mesophyll Materials and Methods The leaves of C loulIlalher/sii are dorsi ventral with th e meso­ Live material of C. Iriphyllum was co llected in natural grassland in phyll distinctly divided into palisade and spongy parenchyma the P r~toria National Botanical Garden and of C. IOllwalbertsii at (Figure I A). In C. triphyllum the mesophyll is more homogene­ Me intjieskop. Pretori a. Voucher specimens were donated to the ous, not clearly divided into palisade and spongy parenchyma National Herbarium, Pretoria (PRE). Material was fixed in FAA (Figure IB), The mesophyll of C. triphyllllm is fairly tightly (Johansen 1940) or O. I M phosphate buITered solution (pH 7.4) of packed, with fewer intercellular spaces th an in C loulIlalbertsii. 5% formaldehyde (Collins & MacNichol 1978); 0.5% caffeine was Numerous substomatal air chambers are present below the adax­ added to the butTered solution, according to the method of Mueller ial and abaxial epidermis of both species. In cross sectio n the and Greenwood (1977). Pieces from the middle of the leaf were leaves of C loulllalbertsii seem thinner th an those of C Iriphyl­ embedded in wax and sectioned at 7 to 10 ).lm on a rotary micro­ lum. ColJenchyma is present at the leaf margin of both C triphyl­ tome. Pieces were also dehydrated, infiltrated and embedded in gly­ hun and C louwalbertsii. col methacrylate (GMA) (Feder & O'Brien 1968) and sectioned at 2- 3 ).tm on a Jung PM 2045 mic rotome. Wax sections were stained Venation in safranin and fast green, whi le GMA sections were stained with the The main vei n in the leaves of both species is raised abaxiaJly. pc:ri odi c a..:id/Schi fT' s (PAS) reaction and counter-stained with tolui­ Collenchymatous tissue is present below the adaxial and abaxial dine blue (Feder & O'Brien 1968). The sections were studied wi th epidermis above an d below the main vein in both species (Figure an Olympus Vanox-S microscope and photographed using I1ford 1F). This coll enchymatous tissue is separated from the main Panf black-and-white film (ASA 50). Leaf epidermis was obtained veins by thin-wall ed isodiametric cell s and a thin-walled bundle by maceration in Jeffrey 's so lution (Kiger 1971 ); il was then stained sheath. In cross secti on the main veins of both species are in a 1% aqueous safranin solution and mounted in Entallen. Pieces arc-shaped with two or three additional vascular bundles adaxi­ of dried lear material were sputter-coated with gold and studied ally. The main vein of C. friphy/lum is sometimes cylind rical, under an lSI SX 25 Scanning Electron Microscope (SE M). with no additional adaxial vascular bundl es. The first-order veins of both species are surrounded by a thin-walled bundle sheath . Observations Sometimes a few collenchymatous cell s occur adaxially of th e Light microscopy first-order veins of C louwalberlsii below the epidermis, but Epidermis they are not continuous with the bundle sheath. Some collenchy­ The epidermal cell s are quadrangular to rectangular in transverse matous cells sometimes occur abaxially of the first-order veins of section. Above and below the main vein the cells are smaller C lriphy/llitn below the epidermis, not continuous with the than over the rest of the leaf. The leaves are amphistomatic bundle sheath, s. Afr. J. BDl. 1998, 64(4) 247

Figure Light micrographs of th e leaf anatomy of the two Clerodendrllnl species. A. Cross section through the leaf of Clerodendrum /Oll­ walberlsii to illustrate the stomata (S) in both the adaxial and abaxial epidermis and the clearly divided palisade (P) and spongy (S P) paren­ chyma of the mesophyJl . Scale bar = 100 Jlm . B. Cross section through the lear of C. triphyllum to illustrate the stomata (S) in both the adaxial and abaxial epidermis and the homogeneous rnesophyll (M). Scale bar = I 00 ~l m . C. Epidermal preparation of C. /oll walberlsii to illu strate the diaeyt! c stomata (5) and irregular intercostal epidermal cells . Scale bar = 100 ).lm. D. Cross section through a pcltate hair in the epidermis of C. louwalbertsii with basal cell (F), uni cellul ar stalk (U) and multicellular head (H); (S) stoma. Scale bar = 50 ~m . E. Epide rm al prepara­ tion of C. iouwaibertsii to illustrate the 8-celled head of the peltate hair. Scale bar = 50 j.lm. F. Cross section through the ma in vein area of C lriphyiium 10 ill ustrate the coll enchyma (e) below the adaxial and abaxial epidermis and the thin walled ti ssue (T) aroun d the vascular bun­ dle. Scale har ~ I 00 ~m. 24X s. A Ii·. 1. Bot. 199X. 64( 4)

Scanning Electron Microscopy (SEM) especially the lower surface and the stomata, to separate species In surface view the leaf surface of C louH'alherlsii shows a and infraspecific taxa. In the present study, significant differ­ bulgy, undulate pattern (Figure 2A). The leaf surface of C. tr;­ ences were observed under the SEM to support the recognition of phyllllnl seems less bulgy and smoother (Figure 2B). In both spe­ two separate taxa: the smooth, bulgy, undulate leaf surface of C cies the leaf margin is recurved and the surface along the margins I01(walherlsii and the reticulate, less bulgy leaf surface of C. tri­ has an elongated pattern as above the main vein, especially obvi­ phy/lurn. ous on the abaxial surface (Figure 2C). The peltate hairs are Solereder (1908), Metcalfe and Chalk (1950), Metcalfe (1979) clearly visible under the SEM (Figure 2D). Under high magnifi­ and Metcalfe and Chalk (1979) reported homogeneous meso­ cation, the surface of C /oltH'alberlsii is quite smooth (Figure phylJ and dorsiventral leaves in the family Verbenaceae. In C 2£) except for occasional striations at the base of the peltate hairs /vulVa/bertsi; the leaves are dorsiventral, but in C. lriphyl/um the and occasionally around the stomata. In C rriphyllum the surface mesophyll is homogeneous. However, it was surprising to find has a 'dry mud' (reticulate) appearance (Figure 2F). No signifi­ two different types of mesophyll arrangement in two species of cant differences were observed in the stomata and peltate hairs of the same genus with similar habitats (grassland) and life forms the two species. (perennial herbs), as observed in this study. Several authors have described the peItate hairs found in many Discussion members of the family Verbenaceae (Solereder 1908, Metcalfe Kereszty (1993- 94) studied the leaves of various Clerodendrum & Chalk 1950, Inal11dar 1969, Theobald el al. 1979, Abu-Asab & species under the SEM and found significant differences, in Cantina 1987, Cantina 1990 & Kereszty 1993- 94). The general

Figure 2 Scanning electron micrographs of the leaf surfaces orthe two Clerodendrum species. A. Adaxial leaf surface orc /oulValbertSll to illustrate the bulgy, undulate pattern, x 210. B. Adaxial leaf surface orc triphyflum to illustrate the less bulgy pattern, x 210. C. Abaxial lear surface of C /ouwalhertsii to illustrate the elongated pattern along the main vein, x 137.4. D. Adaxial leaf surface of C /oulVa/her/sii to illustrate the 8-headed peltatc hair, x 726. E. The smooth abaxial !caf surface of C. louwalbertsii under high magnification, x 1380. F. Abax­ ial leaf surface of C triphyllllm to illustrate the 'dry mud' (reticulate) appearance under high magnitication. x 1380. s. Afr. J. Bot. 1998,64(4) 249

morphology of the peltate hairs present in the two species studied leaf remains. Bot. Rev. 40: 1- 157. was basically the same as that described by these authors, and no FEDER. N. & O'BRIEN, T.P . 1968. Plant microtechnique: some princi­ significant differences were observed between the peltate hairs ples and new methods. Am J. Bot. 55: 123-142. of the two species: in both cases the hairs had an 8-ceJled head, a HERMAN, P.PJ. 1993. Verbcll<'lccac. In: Plants of southern AIIl ca: unicellular stalk and a base cell. names and distribution. (cds) T.H. Arnold & B.c. De WeI. Mem. Bal. Surv, S.I1. 62: 601--604. The stomata in the amphistomatic leaves of Clerodendrum fri­ HERMAN, P.P.). \995. Vcrbcnaceae. A new species in the genus Cicl'O­ phyllum and C louwalbertsij are diacytic, anisocytic, anomo­ del/drum. BOlhaJia 25: 100-·102. eytie and paracytic according to the classification system of INAMDAR. lA. 1969. Epidermal stnlctUfc and ontogeny of stomata in Dilcher (1974) and Wilkonson (1979). Among the Cuban Clero­ some Verbenaceae. Ann BOl. 33: 55- 66. elendrum taxa, Kereszty (1993- 94) found only hypostomatic JOHANSEN, D.A. 1940, Plant microtcchnique. McGraw·Hill. New leaves and anomocytic, actinocytic, anisocytic and paracytic sto­ York. mata. Inamdar (1969) also found only hypostomatic leaves but KERESZTY, Z. 1993-94. SEM analysis on the lear epidermis of'tlle mostly diacytic stomata in the Indian Clerodendrum taxa studied Cuban Clel'Odendl'ulIl taxa (Verhenaceae). Acta BOI. Hungaria 38: by hi m. Catltitlo (1990) reported 3-celled diallelocytic stomata in 353- 368. al l the species of Clerodendrum subgenus Cyclonema he exam­ KIGER, R.W. 1971. Epidennal and cuticu lar mounts of plant material ined . C. Iriphyl/wn and C. lomvalbertsii both bel ong to the sub­ obt ... ined by maceration. Stain Technology 46(2): 71 - 75. genus Cyclonema (Verdcourt 1992) but only diacytic, anisocytic, METCALFE, C.R. 1979. The leaf: ge neral topography and ontogeny of anornocytic and paracytic stomata were observed during the the tissues. In: Anatomy orthe dicotyledons, (cds) C.R. Metcalfe & L. Chalk. 2nd edn. Vol. I: 63-75, 206. Clarendon Press, Oxford. present study. METCALFE, C.R. & CHALK. L. 1950. Anatomy of the di cotyledons Vol. 2. Clarendon Press, Oxford. Conclusion METCALFE, C.R. & CHALK, L. 1979. Anatomy of the dicotyledons. Signifi cant differences observed in leaf surface st ructure as seen 2nd edn. Vol. I. Clarendon Press, Oxford. under the SEM and mesophyll structure, support the recognition MOLDENKE, I-I.N. 1973 . Family 168. Verbenaceae. In : Flora or Pan­ of two distinct species. C Iriphyl/llm and C IOlllvalherlsii. ama, Part 9, (cds) R.E. Woodson, R.W. Schery and Collaborators. Ann Miss. Bot. Gdn60: 4 1- 148. Acknowledgements MUELLER, W.C & GREENWOOD, A.D. t 977. The uttrastructure of Ms C. Steytl and Mr C. Makgakga are thanked for preparation of phenolic cells fi xed with caffeine. J. expo Bal. 29: 757- 764. RETIEF, E, & HERMAN, P.P.J . 1997. Plants of the northern provinces microscope slides. Dr E. Steyn is thanked for assistance with of South Africa: keys and diagnostic characters. Streiilzia 6. procedures and she and Mrs E. du Plessis are thanked for criti­ RUEDA, R.M. 1993. The genus Clerodendrwn (Vcrhenaceac) in Mes­ cally reading the manuscript. The technical support of Dr S. Per­ oamerica. Ann. Miss. Bot. Gdn 80: 870-890. old and Mrs A. Romanowski is gratefull y acknowledged. SOLEREDER, H. 1908. Systematic analomy of the dicotyledons. Oxford , Clarendon Press. References THEOBALD. W.L., KRAHULlK, J. L. & ROLLINS, R.C . 1979. Tri­ ABU-ASA B, M. & CANTINO, P.O. 1987. Phylogenetic implications of chome description and classification. In: Anatomy of the dicotyle­ leaf anatomy in subtribe McJittidinae (Labiatae) and rdatcd taxa. J! dons, (eds.) CR. Metcalre & L. Chalk. 2nd edn. Vol. I: 40- 53,196. Am. Arb. 68: 1-34. Clarendon Press, Oxford. CANTINO, P.O. 1990. The phylogenetic significance of stomata and tri­ VERDCOURT, R 1992. Verbcnaceae. Flora of Tropical East Africa. chomes in the Labiatae and Verb~naceae. J! Am. Arbor. 71: 323-370. A.A. l3alkema. Rotterdam. CO LUNS. B.A . & MACNICHOL, E.F. Uun.) 1978. Long tcnn lixati on WILKONSON, H.P. 1979. The plant surface. Part 1: SlOmata. In : Anat­ for histological studies. Microsc. ACla 81 : 155- 158. omy of the dicotyledons, (eds.) C. R. Metcalfe & L. Chalk. 2nd edn. DILCHER, D.L. 1974, Approaches to the identification of' angiospenn Vol. I: 97-117. Clarendon Press. Oxford.