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Comparative anatomical studies in five southern African species of Crassula: II. Structure of the leaf and the occurrence of transfer cells
S. Afr. J. Bot. 1997.63(2): 95-99
95
Comparative anatomical studies in five southern African species of Crassllla: II. Structure of the leaf and the occurrence of transfer cells A. Moteetee and C.R . Nagendran* Biology Department, National University of Lesoth o, P. O. Roma 180, Lesotho. Rec:eived
23 AI/Ruoff 1996: reviud 13 Jafluary 1997
Crassula dependens Bolus, C. inanis Thunb., C. lancee/ala End l., C. natalensis Schon I. and C. pep/c ides Harv. were collected in Lesotho and South Africa and their anatomical features studied. The leaves of Crassula are typically fleshy, ovoid to elliptic. The unHacial mesaphyll consists of large rou nd, thin-walled, ch lorophy ll-containing parenchyma cells. The palisade tissue is absent. The stomata are always surrounded by a girdle of three subsidiary cells, except in the aquatic taxon, C. inanis, in which vestigial stomata are surrounded by two subs idiary cells and the pore often fails to develop. Electron microscopy revea ls the occurrence of transfe r cells not previously recorded for the genus. Minor leaf vein transfer cells of the B~ty pe occur in all the five species of Crassula. Slem transfe r ce lls associated wit h the phloem occur in C. dependens. Keywords : Crassula, Crassu laceae , epHhem , hydathodes, tannin , venation. ' To whom correspondence should be addressed.
Introduction Many pla nts possess apertures on th eir leaf surfaces through whic h fluid is sl!crcted. Kean ( 1924) indicatl!d that hydat hodes only secrete ami do not absorb water. However, it has been reported that margin al epithem hydathodes may absorb wnter (Wilkinson 1979). Hydathodes occur predominantly on toothed margins of herbs, deciduous shrubs and tree s. Hydathodes in the Crassu laceae have been reported in species of Bryophylll/Jl1 Sali sh. , C((Jssllla L., Ka Lallchoe Ad ans ., Sedam L. and Umbilicus DC. (Metcalfe & Chalk 1950). The di stri buti on of hydat hodcs varies in different species. They may be scattered on both surfaces of the leaf as in Crassilla cultrata L. and C. tetragolltl L., or they may be on the upper surface only as in C. cordata Ail., C. schmidtii Reger and C. peiforara L. (Solcrcder 1908; Kean, 1924). On the other hand they may form rows near leaf margins either on both surfaces as in C. lactea Ail. , or on the lower surfacl! on ly as in C. Lycopodioides Lam and C. sp alll/ata Thunb. (So lereder 1908). In C. argenteel Thunh" the hydathodcs occur over the whole ti ppe r surface and some of them arc arranged in an irregul ar row nl!ar thl! leaf margin (Ross 1969). Hydathodes vary in the number of modified stomata assoc iated with the epithem. In C. /actea, as many as 25 sto mata have been obse rved, while in C. mxet!lea e ight to ten have been observed (Ross 1969); in C. pelforara. Rochea coccinea L. and C. fa/caw Wendl. there is only one relati ve ly large stoma associated with an cpi them (Solereder. 1908). The prese nce of hydathodes is a characrer ortaxollo mi c importance (To lken 1977). Th e lea ves of Cmssula are obovate {Q elliptic (To lken 19S5). The ce ll s th at surround the guard cells of the stomata ex hi bit a characteristic arrangement. Most common ly there is a girdle of threl! subs id iary cells surrounding the guard cells. of which one is smaller than the oth er two. Such stomata are referred to as anisocylic (Wilkinson 1979). The mesophyll is unifaci al. Th e ce lls are thin-wa ll ed and parenChymatous. T he venation of the leaws differs from the typical reti culate venation of dicotyledonous leaves. [n BI)'Ophy/lwll caficymul1 Sali sb., another me mber of the Crassulaceae, the venatio n of the leaf is of the pinnate lype (Yarbrough 1934). In the Crassulaceae three genera have bee n li sted (Q have minor leaf vein transfer cells (Pate & Gu nning 1969). Stem transfer ce lls have been recorded in the genus Sedlllll by Gunning ef
al. (1970). Si nce systematic anatomical studi es have not been carried out on Crassu loideae in ge neral and Crasslila in particular. the present study was undertaken.
Materials and Methods MalUr!.! tlO\wring plant specimens were identifi ed at the Royal Botanic Gardens. Kew. Details of the localities of the specimens used for anato mi ca l and ultrast ructural studi es are in Table I of Motl!etee and Nagendran ( 1997). All plants investigated were collected from Lesotho and South Africa. Voucher specimens have been deposited at the ROML herbarium. The leaves were fixed in 3% gluteraldehyde and post-fixed in 10% Osmi um tetroxide in 0.05 M sod ium cacodyl:Jte buffer at 20°C. Dehydration W:JS done wi th !.!thanol and infiltrat ion and embedding were carried out using spu rr resin (S purr 1969) with propylene ox ide :JS a diluent For light microscopy i-Ilm secti ons were cut usi ng a microtome and stained with 1% toluidine blue, and microscopic studies were carried out usi ng a Leitz Ortholux ph olomi croscope fi tted with differential interference contrast optics. For electron microscopy ultra-t hin sections were collected on Formvar-coated copper grids and stai ned with uranyl acetate and lead ci trate. The sect ions were observed with the l EM-IOOS transmisson electron microscope. To obse rve leaf surfaces and venat ion patterns leaves wcre boiled in absolute alcohol and mounted in lactic acid. Results In th e investigated species of Crassllia leaves are mostly ovate . In C. lla/alellsis and C. pepLuides there are blunt, thick- wa lled hairs on the leaf margin s (Figure lA). The venati on paltern is eit her reti culate as ill C. dependem' (Figu re IB) and C. peploides or pinnate as in C. !allceo/ala and C. illallis. The hydathodcs occur al ong the margins (Figure 1A). In C. depelldens the base of eac h hydathode is associated wit h vascu lar tissue contain ing five or more tracheary elemenL>; ending blindly be low an ep it hem. [n C. IIalaiellsis the hydathodes are prese nt at regular intervals along the marginal veins and are sheathed by tannin-containing cells. In C. Lan ceo/ala there is no evidence of hydathode s but the tracheary elements splay out at the vein endings. Hydat hodes are not readil y discernible in C. peploides and C. inallis except that, in the latter species, there is a group of small parenchyma cell s outside the vein endings (Figure 1C). The stomata are surrou nded by a g irdle of three subsidiary
S. Afr. J. Bot. 1997.63(2)
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Figure J L~ar stru clUre in Crassu/a species. A. C. flataletlSis: surface vie\v of a portion o f leaf showing hairs; H de notes a hydathode. x20; B. C. dependens: su rface view of a portion o f leaf showi ng venation pattern . xl 5; C. C. ilUUlis: part of:i cleared leaF showing lateral veins ending'in a group of thin-walled cells. x520; D. C. defJelldells: su rface view of leaf epidermis showing stom:lta, x230; E. C. iI/aI/is: surface vicw of a stoma with two subsidiary cell s. x400: F. C. peploides: stomata (S) in adaxial epidermis (EP) coaled with cU(ci ie (C), x220.
cells in all species exce pt C. j1lan;s (Figure tD, E). 1n C. illallis stomata an! surrou nded by two subsid iary ce lls (Figun! I E). In tran sverse sec ti on. Ihe sto mata are eve n wi th (he e pidt!rmi s in all the spec ies exa mined (Figu re IF). The mcsoph yll consists of large al most spherica l cells and the intercell ular spaces are re latively sma ll. The xylem in the midrib and minor ve ins co nsists mainly of Iracheary cle ments and the phl oem consists of one or more groups of smaller ce ll s (Fi gure 2A). In C. Il(ffaiellsis the phloem co nsis ts of two or more groups of up to 30 cel ls in the midrib and one group in the minor veins. Tannin does not occ ur in Ihe leaves of C. /anceo/ata. bu t the distribu tion va ri es in the other spcdes. It is mainl y located in the epidermis and bundle sheath (Figures 2B. C). In C. depelldells and C. il/(lllis. tannin also occu rs in th l! mesophyll ce ll s (Figure 2C). Tannin is sparsely
distributed in the mcso phyll cc:lis o r C. pcp/aides. The sieve elements are thin-walled and ovo id in cross-sec tion (Figure 3A). In the minor lea f veins, B-type tra nsfer cell s (Pate & Gu nning 1969) are assoc iated with the sieve ele me nts (Figure 3B). These transfe r cell s conta in large vacuo les and miloc hondria (Figures 3C, D).
Discussion Leaves of the tive spec ies or Crass//{a studied lack palisade tissue but cons ist mainly of spongy mcsophyll , a feature of xerophytic leaves. Although these cell s o r the spongy mesophy ll are loose ly arranged. thl.! interce llul ar spaces arc relatively small. These ce lls contain large vac uoles and c hloroplasts. They are lyp ical of CAM- (Crassu laccan Add Metabolism) performing
S. Afr. J. Bot. 1997,63(2)
97
Figure 2 Leaf structure in CrasslIla species. A. C. dp/wlldem : minor vei n with phloem tissue (P) and vessel elements (XV) in a bundle sheath (85). x990: B. C. depelfdells: transverse secti on of p.w of mesophyll region: note tnnnin cells (T) and vascular bundles (VB). x200: C. C. i/ItI/lis: tannin-containing cells in the abax:ial epidl!rmis and part of the mesophyll with vascular bundle (V B), x590.
cells, that is. large, thin-walled, round cells wi th large vacuoles (Kluge & Ting 1977).
Types and distribution of stomata In all species of Crassu/a examined. except in C. ill allis. the stomata are surrounded by three subsidiary celis, and they ex hibit a characteri stic arrangement. The stomata arise by divisions resembling those of an apical cell ; successive walls are arranged in th ree directions, as seen in su rface view, and the initial cell becomes divided into a girdle of subsidiary ce ll s which encircles the mother cell of the guard cells (Solereder 1908). No other type of stomatal arrangement has been reported in this family, although Wilkinson (1979) has indicated that helicocytic stomata may occur rarely in the Crassulaceae. In the aquatic C. if/aI/is. the number of stom ata is much reduced. This feature is not surprising. for the complete absence of stomnta has been reported in the submerged leaves of so me members belonging to the Droseraceae, the Nymphaeaceae, and the Ranunculaceae (Eames &
MacDaniels 1925). A low frequency of stomata has also been reported in so me hydrophytcs. Unlike other taxa, stomata of C. illallis are oft en onl y surrounded by two subsidiary cells instead of three. Final division docs not occur in some cases and in olht!rs pores faillo develop. The latter arc possibly vestigial stOmata as observed in other hydrophytcs (Eames & MacDaniels 1925).
Venation pattern The leaves of CmsslIia display considerable di versity in their venation syste ms. The simplest syste m is in C. inal1is and it consists of a midrib and two to thrct! branches of smaller veins. In C. iallceo/ala the venation is of a pinnate type, but unlike other dicoty ledons with suc h a sys tem , the seco ndary veins do not terminate at the margins but arc curved backwards. In C. dependellS, the st!condary vei ns arc joined in a series of prominent arc hes and this type of venation is si milar to that described by Hickey (1979) as a ' brochidod romotl s' venati on. A few st!condaries terminate freely in the mcsophyll. In C. peploides the
98
S. Afr. 1. Bot. 1997.63(2)
Figure 3 Leaf ultra~slructurc in Cmssula species. A C dependelIs: phloem parenchyma cells with mitochondria (M), nucelus (N), plastid (P). vacuole (V) and sieve elements (SE); wall ingrowths (pointer) indicate a differentiating transfer cell, x I 0 500; B. C. peploides: sieve elements (SE) and vacuolated (V) transfer cells (TR) containing mitochondria (M) , x5 000; C. C. in(lnis: sieve elements (5E) and transfer cells (TR), x8 000; D. C. illanis: transfer cell (TR) with a prominent central nucleus (N), x 12 600.
venation type is reticulate as in C. dependens but the secondary veins form two sets of arches on each side of the main vein. The more complex system in C. /latalensis is similar to the one described by Hickey (1979) as 'reticulodromous'. The minor veins branch irregularly from the main vein. These veins also join lOgether to form a series of small submarginal arches. There are no free vein endings. In C. argelltea, there are no free vein endings because all veins end in hydathodes.
Ross (\969) observed that hydathodes in a xerophytic taxon, Crassuia argentea, function throughout the life of the plant, whereas in some other plants, such as Caltha palustris and Ranuflcuius jluitans (hydrophyte), hydathodes function only for a short period of time. He argues that in C. argentea, the hydathodes are 'different anatomically and possibly more active physioIogicaIIy'. One might therefore postulate that the hydathodes of other xerophytic and even mesophytic Crassuln may have a similar function .
Hydathodes Hydathodes have been reported in many plants from a wide range of habitats (Ross 1969). In the Crassulaceae they have been reported in species of BJ)'oph.vLlllln, CrassII!a, Kalallchoe, Sedum and Umbilicus (Metcalfe & Chalk 1950). In C inallis the minor veins terminate into a group of small thin-walled parenchyma cells in the mesophyIL Whether these cells constitute a hydathode epithem is difficult to determine, but hydathodes have been reported in some submerged leaves of other dicotyledonous species like Rmwnculus jluitalls Lam and Caltha paillstris L. (Wilkinson 1979). These structures in C (nanis are possibly vestigial hydathodes as they are different from other hydathodes observed in mesophytic species of Crassllla.
Transfer cells It has been well documented that transfer cells occur widely in the plant kingdom (Gunning & Pate 1969; Pate & Gunning 1969). However, because of the vast number of plants in which transfer cells occur, the literature on their occurrence in individual species is scarce. In some species of Crassuia. transfer cells occur in two locations: minor leaf veins and vascular tissue of the stem. Minor leaf vein transfer cells have been found to occur in all the species of Crassula examined. As in other leaves, these cells have a highly polarized distribution of wall ingrowths; they have been designated as B-lype cells (Pate & Gunning 1969). In the Crassulaceae. stem transfer cells have been reported in
s.
Afr. J. Bo1. 1997. 63(2)
Sedll111 (G unning et aL. 1970). In the species of Crassllia examined. stem tran sfe r ce lls have bl!cn fou nd to occu r in the phloem of C. depclldclIs. So me.! of the fa mili es li sted by Gunning el al. (1970). whic h have stem vascular tissue transfer cells. appear also to possess lra nsfl!f cells in minur leaf veins. These include the.! Crassu laccae. Dipsu<.:uceae. Geraniaceae, Loasaceae, Ruhiaccae, etc., but the authors s tated that 'it is quite clear that eVO luti o nary co nditi ons under which stem transfer ce ll s arose did not necessarily prompt a co rresponding development in leaves'. In Crassu/a at least one species , C. dcpcndcfls, has bct!11 found to have bot h m inor vein and ste m vascular transfer cells. Although only a few spcdes have been investigated in the present study. it is clear that mem bl!rs of the ge nus CrassIlla have a combination of distin ct charactl:!rs like the presence of un ifac ial mesophy ll, hydathodes, minor lea f vein trans fer cells and anisocytic stomata. Any further work should incl ude a survey o f more s pecies of Cmssula and indeed of o ther genera of the family, to determine whether the se features arc ubiquitous. In ru ture stu di es. it wou ld be interesting to explore the rdationship between habitat diversity and leaf anatom y.
Acknowledgements This project was tinanced by the aDA through the British Counc il to the GoVt.:rnment of Leso th o . Th is wo rk is part of an M.Phil thesis of one of us (A.M.) approved hy the University of London under the g uid ance of Prof. l.G . Du ckett. T hi s publ icati on was made possible by the SOllthern African B otanical Diversity Ne two rk (SA BON ET) Ihrough support provided by Ihe Regional Centre for Southern Africa, Gaborone, Botswana, US Agency for International Deve lo pment (Pl ot No. 14818 Lebat lane Road, Gaborone West, Extension 6, Gaborone), under the terms o f Grant No. 690-0283-A-OO-5950. The opinions expressed herein arc those of the authors and do not necessarily reflect lhe vi ews of the US Agency for Inh::rnatio nal Development or the SABONET Steering Committee.
99 GUNN ING, B.E.S. & PATE. 1.S. 1969. Transfer cells'. Plant cells with wall ingrowths. specialized in relat ion to short distance transport of so lutes - theIr occurrence, stru cture and developmen t Prolopia.wlla 68 : 107-133. GUNNING. B.E.S .. PATE. J.S. & GREEN, L.W. 1970. Transfe r cells in va scu lar system of stems: Taxono my, associati on with nodcs and structure. Pmlopiamltl7 1: 147- 177. HICKEY. J.L. 1979. A rev ised claSSIfication of the arch itccture of dicotyledo nous leaves. In : Anatomy of Dicotyledons, ed. CR . Metca lfe & L. Chalk. 2nd cdn . Vall. Systcmatic anatomy of leaf and stem. with a brief histo ry of the subject. pp. 25-39. C larendon Press, Ox ford. KEAN . C.H . 1924 . The morph ology and physiology of the leaves of some Crassulaceae. Tram. Pmc. bOI. SOl;. Edil/b. 29: 96-104. KLUGE, K. & TING , LP. 1977. Crassulucean acid metaboli sm: Analysis of an ecolog ical adaptation. Springer-Verlag. Berlin. METCALFE, C.R. & CHALK. L. 1950. Crassulaceae. In: Anatomy of Dicotyledons. Vol. 1. pp. 578-58 1. Clarendon Press, Oxford . MOTEETEE. A. & NAGENDRAN, C.R. 1997 . Comparative anatomical studies in fi ve sou thern African spec ies of Cm.~.wl(l; I. Struc ture of the stem and the root. S. A(r. 1. HOI. 63: 90-94 . PATE, 1.S. & GUNNI NG , B.E.S. 1969. Vascu lar transfer cell s in angiosperm leaves. A taxono mic and morphological survey. Pmtoplasma 68: 135- 156. ROSS. TL. 1969. Vascu lar pattern and hydathodes in leaves of Cra.uula argellfea (Crassulaceae). BOT. Gaz. 130: 267- 270. SOLEREDER. H. 1908. System atic anatomy of the dicotyledons (translated by L.A. Boodle & EE. Fritsch. revised by D.H. Scott) . Clarendon Press. Oxford. SPURR. A.R . 1969. A low viscosity epoxy res in embedding medIUm for e lectron microscopy. 1. Itlt. ReJ. 26 : 3 1-43. TOLKEN . H.R. 1977. A rev isio n of the genus Crassula in southern Afr ica. CIJ/J/r. BoluJ H erb. Rondebo.f(;h 8: 75- 229. TOLKEN. H .R. 1985. Crassuiaccnc. In : Flora of southern Africa 14. ed . O.A . Leistner. pp. 1-244. Bot. Res. In st., South Africa.
References
WILKINSON , H.P. 1979. The plant strucure. In: Anatomy of di coty ledons. ed . C.R. Mctcalfe & L. Chalk, 2nd edn. Vol. I. Systematic anatomy of the leaf and stem. with a bri ef history of the subject, pp. 97165. Clare ndon Press. Oxford.
EAM ES. A.J. & MAC DA NIELS , L.H . 1925. An introdu ction to plant anaLOmy. McG raw- Hill. New York.
YARBROUG H, I .A. 1934. Hi stology of leaf in Bryophylfltm cof),cil1ulll. Am. 1. BOI. 21: 467-482.
95
Comparative anatomical studies in five southern African species of Crassllla: II. Structure of the leaf and the occurrence of transfer cells A. Moteetee and C.R . Nagendran* Biology Department, National University of Lesoth o, P. O. Roma 180, Lesotho. Rec:eived
23 AI/Ruoff 1996: reviud 13 Jafluary 1997
Crassula dependens Bolus, C. inanis Thunb., C. lancee/ala End l., C. natalensis Schon I. and C. pep/c ides Harv. were collected in Lesotho and South Africa and their anatomical features studied. The leaves of Crassula are typically fleshy, ovoid to elliptic. The unHacial mesaphyll consists of large rou nd, thin-walled, ch lorophy ll-containing parenchyma cells. The palisade tissue is absent. The stomata are always surrounded by a girdle of three subsidiary cells, except in the aquatic taxon, C. inanis, in which vestigial stomata are surrounded by two subs idiary cells and the pore often fails to develop. Electron microscopy revea ls the occurrence of transfe r cells not previously recorded for the genus. Minor leaf vein transfer cells of the B~ty pe occur in all the five species of Crassula. Slem transfe r ce lls associated wit h the phloem occur in C. dependens. Keywords : Crassula, Crassu laceae , epHhem , hydathodes, tannin , venation. ' To whom correspondence should be addressed.
Introduction Many pla nts possess apertures on th eir leaf surfaces through whic h fluid is sl!crcted. Kean ( 1924) indicatl!d that hydat hodes only secrete ami do not absorb water. However, it has been reported that margin al epithem hydathodes may absorb wnter (Wilkinson 1979). Hydathodes occur predominantly on toothed margins of herbs, deciduous shrubs and tree s. Hydathodes in the Crassu laceae have been reported in species of Bryophylll/Jl1 Sali sh. , C((Jssllla L., Ka Lallchoe Ad ans ., Sedam L. and Umbilicus DC. (Metcalfe & Chalk 1950). The di stri buti on of hydat hodcs varies in different species. They may be scattered on both surfaces of the leaf as in Crassilla cultrata L. and C. tetragolltl L., or they may be on the upper surface only as in C. cordata Ail., C. schmidtii Reger and C. peiforara L. (Solcrcder 1908; Kean, 1924). On the other hand they may form rows near leaf margins either on both surfaces as in C. lactea Ail. , or on the lower surfacl! on ly as in C. Lycopodioides Lam and C. sp alll/ata Thunb. (So lereder 1908). In C. argenteel Thunh" the hydathodcs occur over the whole ti ppe r surface and some of them arc arranged in an irregul ar row nl!ar thl! leaf margin (Ross 1969). Hydathodes vary in the number of modified stomata assoc iated with the epithem. In C. /actea, as many as 25 sto mata have been obse rved, while in C. mxet!lea e ight to ten have been observed (Ross 1969); in C. pelforara. Rochea coccinea L. and C. fa/caw Wendl. there is only one relati ve ly large stoma associated with an cpi them (Solereder. 1908). The prese nce of hydathodes is a characrer ortaxollo mi c importance (To lken 1977). Th e lea ves of Cmssula are obovate {Q elliptic (To lken 19S5). The ce ll s th at surround the guard cells of the stomata ex hi bit a characteristic arrangement. Most common ly there is a girdle of threl! subs id iary cells surrounding the guard cells. of which one is smaller than the oth er two. Such stomata are referred to as anisocylic (Wilkinson 1979). The mesophyll is unifaci al. Th e ce lls are thin-wa ll ed and parenChymatous. T he venation of the leaws differs from the typical reti culate venation of dicotyledonous leaves. [n BI)'Ophy/lwll caficymul1 Sali sb., another me mber of the Crassulaceae, the venatio n of the leaf is of the pinnate lype (Yarbrough 1934). In the Crassulaceae three genera have bee n li sted (Q have minor leaf vein transfer cells (Pate & Gu nning 1969). Stem transfer ce lls have been recorded in the genus Sedlllll by Gunning ef
al. (1970). Si nce systematic anatomical studi es have not been carried out on Crassu loideae in ge neral and Crasslila in particular. the present study was undertaken.
Materials and Methods MalUr!.! tlO\wring plant specimens were identifi ed at the Royal Botanic Gardens. Kew. Details of the localities of the specimens used for anato mi ca l and ultrast ructural studi es are in Table I of Motl!etee and Nagendran ( 1997). All plants investigated were collected from Lesotho and South Africa. Voucher specimens have been deposited at the ROML herbarium. The leaves were fixed in 3% gluteraldehyde and post-fixed in 10% Osmi um tetroxide in 0.05 M sod ium cacodyl:Jte buffer at 20°C. Dehydration W:JS done wi th !.!thanol and infiltrat ion and embedding were carried out using spu rr resin (S purr 1969) with propylene ox ide :JS a diluent For light microscopy i-Ilm secti ons were cut usi ng a microtome and stained with 1% toluidine blue, and microscopic studies were carried out usi ng a Leitz Ortholux ph olomi croscope fi tted with differential interference contrast optics. For electron microscopy ultra-t hin sections were collected on Formvar-coated copper grids and stai ned with uranyl acetate and lead ci trate. The sect ions were observed with the l EM-IOOS transmisson electron microscope. To obse rve leaf surfaces and venat ion patterns leaves wcre boiled in absolute alcohol and mounted in lactic acid. Results In th e investigated species of Crassllia leaves are mostly ovate . In C. lla/alellsis and C. pepLuides there are blunt, thick- wa lled hairs on the leaf margin s (Figure lA). The venati on paltern is eit her reti culate as ill C. dependem' (Figu re IB) and C. peploides or pinnate as in C. !allceo/ala and C. illallis. The hydathodcs occur al ong the margins (Figure 1A). In C. depelldens the base of eac h hydathode is associated wit h vascu lar tissue contain ing five or more tracheary elemenL>; ending blindly be low an ep it hem. [n C. IIalaiellsis the hydathodes are prese nt at regular intervals along the marginal veins and are sheathed by tannin-containing cells. In C. Lan ceo/ala there is no evidence of hydathode s but the tracheary elements splay out at the vein endings. Hydat hodes are not readil y discernible in C. peploides and C. inallis except that, in the latter species, there is a group of small parenchyma cell s outside the vein endings (Figure 1C). The stomata are surrou nded by a g irdle of three subsidiary
S. Afr. J. Bot. 1997.63(2)
p ,-
f
- F I
>
\.
,
Figure J L~ar stru clUre in Crassu/a species. A. C. flataletlSis: surface vie\v of a portion o f leaf showing hairs; H de notes a hydathode. x20; B. C. dependens: su rface view of a portion o f leaf showi ng venation pattern . xl 5; C. C. ilUUlis: part of:i cleared leaF showing lateral veins ending'in a group of thin-walled cells. x520; D. C. defJelldells: su rface view of leaf epidermis showing stom:lta, x230; E. C. iI/aI/is: surface vicw of a stoma with two subsidiary cell s. x400: F. C. peploides: stomata (S) in adaxial epidermis (EP) coaled with cU(ci ie (C), x220.
cells in all species exce pt C. j1lan;s (Figure tD, E). 1n C. illallis stomata an! surrou nded by two subsid iary ce lls (Figun! I E). In tran sverse sec ti on. Ihe sto mata are eve n wi th (he e pidt!rmi s in all the spec ies exa mined (Figu re IF). The mcsoph yll consists of large al most spherica l cells and the intercell ular spaces are re latively sma ll. The xylem in the midrib and minor ve ins co nsists mainly of Iracheary cle ments and the phl oem consists of one or more groups of smaller ce ll s (Fi gure 2A). In C. Il(ffaiellsis the phloem co nsis ts of two or more groups of up to 30 cel ls in the midrib and one group in the minor veins. Tannin does not occ ur in Ihe leaves of C. /anceo/ata. bu t the distribu tion va ri es in the other spcdes. It is mainl y located in the epidermis and bundle sheath (Figures 2B. C). In C. depelldells and C. il/(lllis. tannin also occu rs in th l! mesophyll ce ll s (Figure 2C). Tannin is sparsely
distributed in the mcso phyll cc:lis o r C. pcp/aides. The sieve elements are thin-walled and ovo id in cross-sec tion (Figure 3A). In the minor lea f veins, B-type tra nsfer cell s (Pate & Gu nning 1969) are assoc iated with the sieve ele me nts (Figure 3B). These transfe r cell s conta in large vacuo les and miloc hondria (Figures 3C, D).
Discussion Leaves of the tive spec ies or Crass//{a studied lack palisade tissue but cons ist mainly of spongy mcsophyll , a feature of xerophytic leaves. Although these cell s o r the spongy mesophy ll are loose ly arranged. thl.! interce llul ar spaces arc relatively small. These ce lls contain large vac uoles and c hloroplasts. They are lyp ical of CAM- (Crassu laccan Add Metabolism) performing
S. Afr. J. Bot. 1997,63(2)
97
Figure 2 Leaf structure in CrasslIla species. A. C. dp/wlldem : minor vei n with phloem tissue (P) and vessel elements (XV) in a bundle sheath (85). x990: B. C. depelfdells: transverse secti on of p.w of mesophyll region: note tnnnin cells (T) and vascular bundles (VB). x200: C. C. i/ItI/lis: tannin-containing cells in the abax:ial epidl!rmis and part of the mesophyll with vascular bundle (V B), x590.
cells, that is. large, thin-walled, round cells wi th large vacuoles (Kluge & Ting 1977).
Types and distribution of stomata In all species of Crassu/a examined. except in C. ill allis. the stomata are surrounded by three subsidiary celis, and they ex hibit a characteri stic arrangement. The stomata arise by divisions resembling those of an apical cell ; successive walls are arranged in th ree directions, as seen in su rface view, and the initial cell becomes divided into a girdle of subsidiary ce ll s which encircles the mother cell of the guard cells (Solereder 1908). No other type of stomatal arrangement has been reported in this family, although Wilkinson (1979) has indicated that helicocytic stomata may occur rarely in the Crassulaceae. In the aquatic C. if/aI/is. the number of stom ata is much reduced. This feature is not surprising. for the complete absence of stomnta has been reported in the submerged leaves of so me members belonging to the Droseraceae, the Nymphaeaceae, and the Ranunculaceae (Eames &
MacDaniels 1925). A low frequency of stomata has also been reported in so me hydrophytcs. Unlike other taxa, stomata of C. illallis are oft en onl y surrounded by two subsidiary cells instead of three. Final division docs not occur in some cases and in olht!rs pores faillo develop. The latter arc possibly vestigial stOmata as observed in other hydrophytcs (Eames & MacDaniels 1925).
Venation pattern The leaves of CmsslIia display considerable di versity in their venation syste ms. The simplest syste m is in C. inal1is and it consists of a midrib and two to thrct! branches of smaller veins. In C. iallceo/ala the venation is of a pinnate type, but unlike other dicoty ledons with suc h a sys tem , the seco ndary veins do not terminate at the margins but arc curved backwards. In C. dependellS, the st!condary vei ns arc joined in a series of prominent arc hes and this type of venation is si milar to that described by Hickey (1979) as a ' brochidod romotl s' venati on. A few st!condaries terminate freely in the mcsophyll. In C. peploides the
98
S. Afr. 1. Bot. 1997.63(2)
Figure 3 Leaf ultra~slructurc in Cmssula species. A C dependelIs: phloem parenchyma cells with mitochondria (M), nucelus (N), plastid (P). vacuole (V) and sieve elements (SE); wall ingrowths (pointer) indicate a differentiating transfer cell, x I 0 500; B. C. peploides: sieve elements (SE) and vacuolated (V) transfer cells (TR) containing mitochondria (M) , x5 000; C. C. in(lnis: sieve elements (5E) and transfer cells (TR), x8 000; D. C. illanis: transfer cell (TR) with a prominent central nucleus (N), x 12 600.
venation type is reticulate as in C. dependens but the secondary veins form two sets of arches on each side of the main vein. The more complex system in C. /latalensis is similar to the one described by Hickey (1979) as 'reticulodromous'. The minor veins branch irregularly from the main vein. These veins also join lOgether to form a series of small submarginal arches. There are no free vein endings. In C. argelltea, there are no free vein endings because all veins end in hydathodes.
Ross (\969) observed that hydathodes in a xerophytic taxon, Crassuia argentea, function throughout the life of the plant, whereas in some other plants, such as Caltha palustris and Ranuflcuius jluitans (hydrophyte), hydathodes function only for a short period of time. He argues that in C. argentea, the hydathodes are 'different anatomically and possibly more active physioIogicaIIy'. One might therefore postulate that the hydathodes of other xerophytic and even mesophytic Crassuln may have a similar function .
Hydathodes Hydathodes have been reported in many plants from a wide range of habitats (Ross 1969). In the Crassulaceae they have been reported in species of BJ)'oph.vLlllln, CrassII!a, Kalallchoe, Sedum and Umbilicus (Metcalfe & Chalk 1950). In C inallis the minor veins terminate into a group of small thin-walled parenchyma cells in the mesophyIL Whether these cells constitute a hydathode epithem is difficult to determine, but hydathodes have been reported in some submerged leaves of other dicotyledonous species like Rmwnculus jluitalls Lam and Caltha paillstris L. (Wilkinson 1979). These structures in C (nanis are possibly vestigial hydathodes as they are different from other hydathodes observed in mesophytic species of Crassllla.
Transfer cells It has been well documented that transfer cells occur widely in the plant kingdom (Gunning & Pate 1969; Pate & Gunning 1969). However, because of the vast number of plants in which transfer cells occur, the literature on their occurrence in individual species is scarce. In some species of Crassuia. transfer cells occur in two locations: minor leaf veins and vascular tissue of the stem. Minor leaf vein transfer cells have been found to occur in all the species of Crassula examined. As in other leaves, these cells have a highly polarized distribution of wall ingrowths; they have been designated as B-lype cells (Pate & Gunning 1969). In the Crassulaceae. stem transfer cells have been reported in
s.
Afr. J. Bo1. 1997. 63(2)
Sedll111 (G unning et aL. 1970). In the species of Crassllia examined. stem tran sfe r ce lls have bl!cn fou nd to occu r in the phloem of C. depclldclIs. So me.! of the fa mili es li sted by Gunning el al. (1970). whic h have stem vascular tissue transfer cells. appear also to possess lra nsfl!f cells in minur leaf veins. These include the.! Crassu laccae. Dipsu<.:uceae. Geraniaceae, Loasaceae, Ruhiaccae, etc., but the authors s tated that 'it is quite clear that eVO luti o nary co nditi ons under which stem transfer ce ll s arose did not necessarily prompt a co rresponding development in leaves'. In Crassu/a at least one species , C. dcpcndcfls, has bct!11 found to have bot h m inor vein and ste m vascular transfer cells. Although only a few spcdes have been investigated in the present study. it is clear that mem bl!rs of the ge nus CrassIlla have a combination of distin ct charactl:!rs like the presence of un ifac ial mesophy ll, hydathodes, minor lea f vein trans fer cells and anisocytic stomata. Any further work should incl ude a survey o f more s pecies of Cmssula and indeed of o ther genera of the family, to determine whether the se features arc ubiquitous. In ru ture stu di es. it wou ld be interesting to explore the rdationship between habitat diversity and leaf anatom y.
Acknowledgements This project was tinanced by the aDA through the British Counc il to the GoVt.:rnment of Leso th o . Th is wo rk is part of an M.Phil thesis of one of us (A.M.) approved hy the University of London under the g uid ance of Prof. l.G . Du ckett. T hi s publ icati on was made possible by the SOllthern African B otanical Diversity Ne two rk (SA BON ET) Ihrough support provided by Ihe Regional Centre for Southern Africa, Gaborone, Botswana, US Agency for International Deve lo pment (Pl ot No. 14818 Lebat lane Road, Gaborone West, Extension 6, Gaborone), under the terms o f Grant No. 690-0283-A-OO-5950. The opinions expressed herein arc those of the authors and do not necessarily reflect lhe vi ews of the US Agency for Inh::rnatio nal Development or the SABONET Steering Committee.
99 GUNN ING, B.E.S. & PATE. 1.S. 1969. Transfer cells'. Plant cells with wall ingrowths. specialized in relat ion to short distance transport of so lutes - theIr occurrence, stru cture and developmen t Prolopia.wlla 68 : 107-133. GUNNING. B.E.S .. PATE. J.S. & GREEN, L.W. 1970. Transfe r cells in va scu lar system of stems: Taxono my, associati on with nodcs and structure. Pmlopiamltl7 1: 147- 177. HICKEY. J.L. 1979. A rev ised claSSIfication of the arch itccture of dicotyledo nous leaves. In : Anatomy of Dicotyledons, ed. CR . Metca lfe & L. Chalk. 2nd cdn . Vall. Systcmatic anatomy of leaf and stem. with a brief histo ry of the subject. pp. 25-39. C larendon Press, Ox ford. KEAN . C.H . 1924 . The morph ology and physiology of the leaves of some Crassulaceae. Tram. Pmc. bOI. SOl;. Edil/b. 29: 96-104. KLUGE, K. & TING , LP. 1977. Crassulucean acid metaboli sm: Analysis of an ecolog ical adaptation. Springer-Verlag. Berlin. METCALFE, C.R. & CHALK. L. 1950. Crassulaceae. In: Anatomy of Dicotyledons. Vol. 1. pp. 578-58 1. Clarendon Press, Oxford . MOTEETEE. A. & NAGENDRAN, C.R. 1997 . Comparative anatomical studies in fi ve sou thern African spec ies of Cm.~.wl(l; I. Struc ture of the stem and the root. S. A(r. 1. HOI. 63: 90-94 . PATE, 1.S. & GUNNI NG , B.E.S. 1969. Vascu lar transfer cell s in angiosperm leaves. A taxono mic and morphological survey. Pmtoplasma 68: 135- 156. ROSS. TL. 1969. Vascu lar pattern and hydathodes in leaves of Cra.uula argellfea (Crassulaceae). BOT. Gaz. 130: 267- 270. SOLEREDER. H. 1908. System atic anatomy of the dicotyledons (translated by L.A. Boodle & EE. Fritsch. revised by D.H. Scott) . Clarendon Press. Oxford. SPURR. A.R . 1969. A low viscosity epoxy res in embedding medIUm for e lectron microscopy. 1. Itlt. ReJ. 26 : 3 1-43. TOLKEN . H.R. 1977. A rev isio n of the genus Crassula in southern Afr ica. CIJ/J/r. BoluJ H erb. Rondebo.f(;h 8: 75- 229. TOLKEN. H .R. 1985. Crassuiaccnc. In : Flora of southern Africa 14. ed . O.A . Leistner. pp. 1-244. Bot. Res. In st., South Africa.
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