Bothalia 15, 3 & 4: 587-590 <1985)

The Kranz syndrome in the Eragrostideae (, ) as indicated by carbon isotopic ratios*

HECTOR O. PANARELLO ** and EVANGELINA SÁNCHEZt

Keywords: carbon isotope ratios, Eragrostideae. Kranz syndrome

ABSTRACT 1!C/'-'C ratios are generally regarded as being very reliable indicators of Q or C4 photosynthesis. These relative carbon isotope ratios are expressed as a negative 6nC and fall into two distinct groups: Kranz (or C4) with Ó between -9°/no and -18°/«u and non-Kranz (C,) plants with 6 between -22°/™ and -28"/no. In this paper, 29 taxa, representing 12 genera, of the tribe Eragrostideae were examined by mass spectrometry for their é'-'C in dried leaf tissue. Ail these taxa proved to be C, plants with 5ISC values ranging between -13,6°/oo and -10.9°/oo. These findings confirmed published leaf anatomical observations which showed that all the studied taxa had characteristic Kranz leaf anatomy.

INTRODUCTION average of -25°/oo (Fig. 1). In the Poaceae, no ra­ tios between -18°/oo and -22°/oo have been re­ A syndrome of anatomical, cytological and phy­ ported. Plants with Crassulacean Acid Metabolism siological characters, all related to aspects of the car­ also have high carbon isotope ratios (Fig. 1), but are bon fixation process, has been reported from several not relevant in this study since CAM has not been families of Angiosperms, This syndrome has been demonstrated in the Poaceae (Brown, 1977). called the Kranz syndrome, C4 photosynthesis, and the Hatch-Slack pathway. Kranz plants exhibit a The determination of carbon isotope ratios is a high degree of efficiency in the utilization of ambient very' convenient method for classifying plants as be­ C 02, resulting in maximum photosynthetic produc­ ing Kranz or non-Kranz. Only small amounts of tion at high temperature and high light intensity. tissue are required, the data are very accurate The anatomical characteristics of this syndrome have and objective and the plant material need not be been known for 100 years (Haberlandt, 1882) but physiologically active. The use of dried specimens the physiological aspects have only been known for from herbarium sheets is, therefore, possible. nearly 20 years (Kortschak et a!., 1965 and Hatch & Slack, 1966). MATERIALS The anatomical specializations of the Kranz syn­ Leaf tissue was taken from herbarium sheets. This drome include the presence of a chlorenchymatous plant material was obtained from many sources, and bundle sheath of large, thick-walled cells containing these are all gratefully acknowledged. Sources in­ specialized chloroplasts distinct from those of the cluded: Dr Thomas R- Soderstrom, US National mesophyll — they are a greater size, a greater num­ Herbarium. Smithsonian Institution, Washington, ber per Kranz cell are present and they accumulate DC (US); Museo Argentino de Ciencias Naturales starch- In addition, the mesophyll cells adjoin the 'Bernardino Rivadavia' (BA); Herbario Gaspar bundle sheath and are radially arranged, Plants pos­ Xuarez, Cátedra de Botánica, Facultad de Agrono- sessing the Kranz syndrome fix carbon initially into mia, Universidad de Buenos Aires (BAA); Instituto four-carbon acids (oxalo-acetic acid, malic acid and de Botánica Darwinion (SI); Instituto Miguel Lillo, aspartic acid) in these mesophyll cells (Hatch & Tucumán (LIL); Instituto de Botánica Agricola, Slack. 1970). INTA, Castelar (BAB); Instituto de Botánica, Fac­ The only way to prove definitely that a plant is ultad de Agronomia, Universidad Nacional del No- Kranz o r non-Kranz is to investigate both its physiol­ reste (CTES). ogy, either directly, or indirectly by means of 13C/12C The genera of the Eragrostideae examined, ar­ ratios, and to examine its anatomy, particularly the ranged alphabetically, are given in Table 1. All leaf anatomy. Differences in carbon isotope ratios voucher specimen sheets from which material was between Kranz and non-Kranz plants are well docu­ taken have been annotated with labels stating the mented (Fritz & Fontes, 1980). 6 I3C values for C4 13C/J-C ratio of that specimen. plants range between -18°/oo and -9°/oo with an average of about -12%o, whereas the 6 I3C for C3 METHODS plants is between -38°/oo and -22°/oo with an About 8 mg of the leaf samples were dried under vacuum at a temperature of llO^C for 8 hours or * INGEIS contribution No. 71 overnight. Every sample was then mixed with 100 ** Instituto de Geocronologia y Geología Istotópica, Ciudad mg vanadium pentoxide (V205) in a quartz vial and Universitaria. Buenos Aires, . flame sealed under vacuum of about 10-4 mbar. The t Museo Argentino de Ciencias Naturales. ‘Bernardino Rivada- vial, containing the sample and the vanadium pen­ via’, Buenos Aires. Argentina. toxide (V205) was allowed to react at 1000°C in an 588 Bothalia 15, 3 & 4 (1985)

mass 45/44 ratio was then determined by measuring 30 the samples in a Micromass 602-D Mass spectrome­ ter. A reference and standard CO, from Carrara

20 - marble was used (Panarello el al., 1980). The results arc expressed as 6 '-'C (%o) defined as follows: 10 - >- Rs O 6 i- 'C lOOO'Voo jn_ T -[ RPDB *HI 0 JE B Where Rs = Isotopic ratio of the sample o RPDB= Isotopic ratio of the STANDARD uj 20 QC PDB (Chicago PDB Standard, Be- lemnitella americana from the Cre­ 10 taceous Pcdee formation. South Carolina JCraig, (1954)). o In. The precision of measurement is ± 0,l()/oo of the ratio. If 6 IJC is > 0 the sample is heavier than 120 the standard, if less than 0 the sample is lighter, or depleted, in comparison with the standard. From this definition 6 IJC PDB = 0. 110 - Carbon occurs in nature as two stable isotopes '-C and 13C, with the following average abundance: 100 12C : 98,89% !3C : 1,11% 90 - However, the l3C/|2C ratio is not constant and undergoes variations of a few parts per thousand in 80 this ratio due to some physical and chemical pro­ cesses. This variation in the isotopic composition is known as ‘isotopic fractionation’. One of the most 70 important processes in nature which causes isotopic fractionation of carbon is photosynthetic carbon as­

60 - similation by green plants. Both terrestrial and marine plants have lower !iC/l2C ratios than their respective carbon sources, 5 0 - atmospheric CO,(0-—7; -8°/oo) and ocean carbon­ ates (6 — °/oci). This means that during photosyn­ 40 - thetic CO-, fixation there is preferential utilization of !2C and exclusion of ,3C (Craig, 1957; Smith & Epstein, 1971). 304 RESULTS 20 The Ó i3C values of the taxa examined in this study are presented in Table 1. These carbon isotope ra­ 10. tios are also compared with published anatomical observations. As always, in the Poaceae, the 6 13C values corroborate the leaf anatomy and all taxa D_ P—a . have C4 carbon isotope ratios (between -I3,6°/oo -3 0 -2 0 -1 0 and -10,9%o) which agree with published ac­ %o&,3C PDB counts of Kranz leaf, and stem, anatomy for these taxa. This study has, therefore, confirmed the pres­ ence of the Kranz syndrome in these selected rep­ FiG. 1. — Carbon isotopic composition of photosynthetically fixed carbon: a. terrestrial plants; b, known C,and C, plants; resentatives of the Eragrostideae. c, known CAM plants. Reproduced from Fritz & Fontes. 1980. ACKNOWLEDGEMENTS The authors are indebted to Dr Enrique Linares for facilities made available at the INGEIS and to electric furnace for five minutes, according to the the staff of the Stable Isotopes Laboratory. Miss technique described by Panarello et al. (1983). Cristina Maetakeda (CONICET) is thanked for technical assistance and Dr R. P. Ellis (Botanical The resultant CO, was introduced into the purifi­ Research Institute, Pretoria) for reading the manu­ cation line by breaking the vial in a vacuum by script. means of a special ‘vial breaker’. Water and light gases were removed and the CO, was transferred to This research was supported with CONICET a sample bottle by trapping with liquid nitrogen. The funds. TABLE 1. —5* values of selected representatives of the Eragrostideae

Species 6 13c%o Kranz leaf anatomy Voucher specimens

; benthamiam (S, Watson) Hackel -10,9 Brown, 1958; Cáccrcs, 1950,51; Metcalfe, 1960; Sanchez, 1983a Soriano 1056 (BAB) bigelovii (S. Watson) Hackel -13,2 Brown, 1977; Sanchez, 1983a J. and Ch. Reeder 5833 (US) hitchcockii Lahitte -11,3 Sanchez, 1983a Castellanos s.n, (BA 33421) kingii (S. Watson) Hackel -12,5 Sanchez, 1983a J. and Ch. Reeder 5292 (US) n aegyptium (L.) K. Richt. -12,7 Brown, 1977; Metcalfe, 1960; Sanchez, 1974 Cabrera etal. 27474 (BAA) lchella (H.B.K.) WUld. ex Rydberg -12,3 Brown, 1977; Sanchez, 1979a, 1983b J, Beatlcy 4886 (US) iomerata (Walter) Burkart -It,6 Sanchez, 1978 Hauman s.n, (BA 38750) (L.) Gaertner -11,8 Brown, 1958, 1977; Sanchez, 1974 W. Partridge s.n. (BA 60697) ■hya (Lam.) Lam. -11,6 Sanchez, 1974 W. Partridge s.n. (BA 59838) ■naceum var. kurtzianum(L.R. Parodi) Anton -13,0 Sanchez, 1979a Kurtz 9850 (BAA) " var. longigtume (L.R. Parodi) Anton -11,7 Sanchez, 1979a A.T. Hunziker 2636 (BA) ” var. pygmaeum (Hackel) Anton -11,8 Sanchez. 1979a Castellanos &.n. (BA 1999S) jsum var. arisrtghimis (Caro) Sanchez -11,7 Sanchez, 1979a Covas 2071 (SI) " var. longearistatum (Kurtz) Anton -12,6 Sanchez, 1979a Castellanos s.n. (BA 28/58) ” var. mendocinum (L.R. Parodi) Nicora -11,6 Sanchez, 1979a Perez-Moreau s.n. (BA 12497) var. parodianum Sanchez -13,5 Sanchez, 1979a Sanchez & Arriaga 1219 (BA) var. pilosum (Buckl.) Nash -12,3 Brown, 1977; Sanchez, 1979a; Smith & Brown, 1973 G. Fisher 43041 (BA) ia (Griseb.) Vasey -12,0 Sanchez, 1975 Castellanos s.n. (BA 24/1471) avensis (Kuntze) Parodi -12,4 Sanchez, 1975 Castellanos s.n, (BA 10231) Phil. -11,3 Sanchez, 1984 De la Sota 224 (LIL) na Griseb. -12,6 Sanchez, 1984 J.H. Hunziker & O. Caso 4064 (BAB) bens Phil. -11,6 Sanchez, 1984 Hunziker & Krapovickas 5887 (BAB) cina Phil. -1 2 ,0 Cáceres, !9S0; Metcalfe, 1960; Sanchez, 1984 Spegazzini s.n. (BAB 28237) tso (N utt.) Torrey -1 2 ,5 Sanchez, 1984 B. Rohrer s.n, (LIL 286743) otam (Michaux) Nicora -12,7 Metcalfe, 1960; Nicora, 1962 H.B. Gephardt 1099 (BA) evifolius Phil. -13,6 Brown, 1977; Cáceres, 1950 C.G. Pringle (BA 9306) Hitchcock -12,2 Brown, 1977; Metcalfe, 1960; Smith& Brown, 1973 W. Benner 10295 (BA) nsis Nees -12,2 Sanchez, 1979a; Smith & Brown, 1973 H. Pueyo 87 (CTES) fus (Nees) Ekman -11,4 Brown, 1958; Brown, 1977 Molfino s.n. (BA 39580) 590 Bothalia 15, 3 & 4 (1985)

UITTREKSEL KORTSCHAK. H.P., HARTT C. E. & BURR. G. O.. 1965. Carbon dioxide fixation in sugar-canc leaves. PI. Physiol. 40: Die ,3d ,2C verhoudings word algemeen as baie be- 209-213. troubare indikaiors van C, o f fotosintetiese roetes METCALFE, C. R.. 1960. Anatomy o f the . I. beskou. Hierdie relatiewe koolstofisotoopverhou- Gramineae. Oxford: Oxford University Press. dings word as negatiewe Ó ,JC uitgedruk en val in twee NICORA. E .. 1962. Revalidación del genero de Grammeas "Nee- dttidelike groepe: Kranz (of C4) plante met tussen ragrostis' de la flora norteamericanu. Revta argent. Agron. -9"I(h> en -I8n/oo en nie-Kranz (of C () plante met 29: 1-11. b tussen -22"loo en -28°ltm. In hierdie artikel is PANARELLO, H. O., GARCIA. C. M.. VALENCIO, S.A. & 29 taksons, wat 12 genera van die tribus Erugrosti- LINARES. E., 1980. Determinación de la composicion deae verteenwoordig, deur mtddel van massaspektro- isotópica del carbono, en carbonatos. su utílización en Hidro- metrie vir hulle bIJC in gedroogde blaarweefsel, be- geologia y Gcologia. Revta Asoc. geol. argent. 35: 460-466, studeer. Dit is bewys dat al hierdie taksons C\plum e PANARELLO. H. O.. ALBERO, M. C. & ANGIOLINI, F. E. is met 6 «C waardes wat varieer tussen -I3,iV/oo en 1983. Stable isotope fractionation during the benzene synthe­ -10,9°/oo. Hierdie bevindinge het gepubliseerde sis for radiocarbon dating. Radiocarbon 25,2: 529-532. anatomiese waarnemings van blare, war getoon het SÁNCHEZ. E.. 1974. Subfamilia Eragrostoideae (Gramineae): Tribu Eragrosteae: Anatomia foliar de las cspecies argenti- dat al die bestudeerde taksons kenmerkende Kranz nas de los generos Eieusine y Dactyloctenittm. Dartviniana blaaranatomie besit, bevestig. 18: 526-538. SÁNCHEZ, E,, 1975. Anatomia foliar de las cspecies REFERENCES del género Gouinia Fournier (Gramineae). Lilloa 34,7: 89-106. BROWN, W. V.. 1958. Leaf anatomy in grass svstentatics, Bot. Gaz. 119.3: 170-178. SÁNCHEZ, E.. 1978. Anatomia foliar de Diandrochloa gloine- rata (Walt.) Burkart (Gramineae). Lilloa 35,1: 67-72. BROWN, W. V., 1977. The Kranz Syndrome and its subtypes in grass systematics. Mem, Torrey hot. Club 23,3: 1-97. SÁNCHEZ. E.. 1979a. Anatomia foliar de las especies y varie- dadcs argentinas de los generos Tridens y Erioneuron (G ra­ CACERES, M. R.. 1950. Los caracteres anatómico-foliares de mineae). Dartviniana 22,1-3: 159-175. Munroa mendocina y Blepharidacltne benthamiana. Revta ar­ gent. Agron. 17: 233-240. SANCHEZ, E.. 1983a. Estudios anatomicos en el genero Blepha- CACERES. M. R ., 1951. La anatomia foliar de Sclerupogon bre- ridachne Hackcl (Poaccac. Eragrostoideae, Eragrosteae). vtfolius y sus relaciones taxonomicas. Revta urgent. Agron. Revta. M2: 115-149. SÁNCHEZ. E., 1983c. Estudios anatomicos en el ginero Munroa Torrcv (Poaceae, Chloridoideae, Eragrostideae). En prensa FRITZ. P. & FONTES. J. C-, [980. A Handbook of Environ­ rcvista Darwniana 25. mental Isotope Geochemistry. Volume I. The terrestrial en­ vironment. SÁNCHEZ, E., 1984. Estudios anatomicos en el genero Munroa (Poaccac, Chloridoideae, Eragrostideae). Darwiniana 25, HABERLANDT, D. G., 1882. Vergleichende Anatomie des as- 1-4: 43-57. similatorischen Gewebesystems der Pflanzen. Pringsheiins Jb. h'«s. Bot. Bd. 13. SMITH. B. N. & EPSTEIN. S., 1971. Two categories of "C /|:C HATCH. M. D. & SLACK, C. R., 1966. Photosynthesis by ratios for higher plants. PI. Physiol. 47; 380-384. sugar-cane leaves. Biochem, J. 101: 103-111, SMITH. B. N. & BROWN, W. V.. 1973. The Kranz syndrome in HATCH, M. D. & SLACK. C. R.. 1970. Photosynthetic CO.fix­ the Gramineae as indicated by carbon isotopic ratios. Am. J, ation pathways. A. Rev. P i Physiol. 21: 141-162. Bot. 60,6: 505-513. Bothalia 15, 3 & 4: 591-5% (1985)

The Rubus in South Africa. I. Chromosome numbers and geographical distribution of species

J. J. SPIES* and H. DU PLESSIS*

Keywords: chromosome numbers, geographical distribution, hybridization, polyploidy, Rubus

ABSTRACT

The geographical distribution of 14 of the Rubus species in South Africa is presented. Chromosome numbers of nine of the species were determined: six for the first time, one is confirmed and additional polyploid levels are described for the other two species. It is demonstrated that the South African species of the subgenus Idaeobatus contain less diploid specimens and more polyploid specimens than their extra-African counterparts. This phenomenon could be attributed to hybridi­ zation between the subgenera Eubatus and Idaeobatus.

INTRODUCTION sults of a preliminary study on chromosome numbers and species distribution of the most important The genus Rubus is spread over all continents and species are presented in this paper. Other papers in is found in most climatic regions. Focke (1910-1914) this series will include studies on meiotic chromo­ divided this genus into 12 subgenera of which only some behaviour, reproduction, hybridization and two are present in South Africa. All the represent­ will be concluded with a cytotaxonomic study of the atives of the subgenus Eubatus Focke are intro­ genus Rubus in South Africa. duced, whereas the subgenus Idaeobatus Focke con­ tains both indigenous and introduced species. MATERIALS AND METHODS Introduced Eubatus species all represent the sub­ The plants used in this study were collected series Suberecti of the series Moriferi and include the throughout South Africa and transplanted under species Rubus afftnis Wh. & N ., R. cuneifolius quarantine in the Pretoria National Botanical Gar­ Pursh., R. pascuus Bailey and R. flagellaris Willd. den. The following 35 plants, representing nine dif­ The introduced Idaeobatus species. R. niveus ferent species, were used: Thunb. and R. phoenicolasius Maxim., form part of the series Nivei of the section Idaeanthi. The in­ digenous Idaeobatus species include the sections Ro­ Eubatus stfolii (R. rosifolius Sm .), Afromontani (R. immixtus Rubus affinis: C.E. Gust.) and Idaeanthi [various species of the se­ TRANSVAAL. — 2329 (Pietersburg): Dap Naude Dam ries Afroidaei, including R. apetalus Poir., R. inter- (-D D ), Stirton 5746. currens C.E. Gust., R. longepedicellatus (C-E. Gust.) C.H. Stirton, R. pinnatus W illd., R. rigidus R. cuneifolius: Sm ., R transvaliensis C,E. Gust, and R. ludwigii NATAL. — 2929 (Underberg): 14 km from Swartberg to Eckl. & Zeyh.. with its subspecies ludwigii and spa- Underberg (-CD), Stirton 8154. 2930 (Pietermaritzburg): 3 km tiosus C.H. Stirton]. For the purpose of this study R. from Midmar Dam to Lions River (-C B ), Henderson & Gaum 93; adolfl-friederici Engl., R ecfclonii Focke and R. ex- 5 km from Pietermaritzburg to Mooi Rjver (-CB), Liengme s.n. succus Steud. are included in R. apetalus Poir. be­ 3029 (Kokstad): 11 km from Harding to Weza (-DB), Stirton 8102. cause integration between these taxa renders the separation into distinct species impossible. Plants R. pascuus: collected from a hybrid swarm in eastern Transvaal are included in R. x proteus C.H. Stirton. TRANSVAAL. — 2430 (Pilgrim's Rest): 1 km from Graskop to Sabie (-DD). Henderson & Gaum 18, Stirton 9800. 9861 & The problems with Rubus in South 9868. Africa are aggravated by the occurrence of apo- mixis, hybridization among indigenous species and R. flagellaris: between indigenous and introduced species, the va­ TRANSVAAL. — 2530 (Lydenburg): Kaapse Hoop (-DB), riation produced by a breeding program and subse­ Henderson & Gaum 2. quent escape from cultivation of those plants and in­ adequate collected herbarium material. The aim of Idaeobatus this study is, therefore, to provide cytogenetical evi­ dence for the species delimitation in the South Afri­ R. apetalus: can species of Rubus. To achieve this goal, the re­ TRANSVAAL. — 2430 (Lydenburg): Kaapse Hoop (-DB), Henderson & Gaum 6. ------NATAL. —2929 (Underberg): Clairmont Plantation (-DD), * Botanical Research Institute, Department of Agriculture & G. Hemm s.n. a & b. 2930 (Pietermaritzburg): Endeni Farm Water Supply. Private Bag X101, Pretoria 0001. (-CC). Wells 5000. 592 Bothalia 15, 3 & 4 (1985)

R. longepedicellatus: Rubus species: TRANSVAAL. — 2430 (Pilgrim s Rest): ! km from Graskop TRANSVAAL. — 2430 (Pilgrim's Rest): 5 km from Graskop to Sabíe (-DD), Henderson <6 Gaum 22. Stirton 9862. 2530 (Ly- to Sabie (-D D ), Henderson A Gaum 24. denburg): 5 km from Lydenburg to Sabie (-AB), Henderson A Specimens are housed in the National Herbarium, Gaum 36; Brooklands (-BA). Henderson A Gaum 14. Pretoria (PRE). Chromosome counts were made R. pinnatus: from meiotic squashes in aceto-carmine (Darlington TRANSVAAL. — 2530 (Lydenburg): Brooklands (-BA). & LaCour, 1976). Between 20 and 25 cells per plant Henderson <6 Gaum 15. were studied. NATAL. — 3029 (Kokstad): 4 km from Kokstad to Weza Distribution maps were obtained by using the data (-C B ), Arnold 1335. o f all Rubus specimens available on PRECIS (Pre­ R. ludwigii: toria computerized information system) (Gibbs Rus­ NATAL. — 2929 (Underberg): Kamsberg Nature Reserve sell & Gonsalves, in press). (-B D ), Henderson A Gaum 41. CAPE. — 3226 (Fort Beaufort): Hogsback (-DB). Admiraal A RESULTS AND DISCUSSION Drijfkoul 2940. All chromosome numbers determined were mul­ R. x proteus: tiples o f 7 and somatic chromosome numbers o f 14, TRANSVAAL. — 2430 (Pilgrim's Rest): Bourkes Luck 21, 28, 35, 42, 49 and 56 were observed in the inves­ (-D B ). Henderson A Gaum 27, 28. 31 A 32; Mac-Mac Falls tigated South African Rubus species (Table 1). Poly­ (-D D ), Henderson A Gaum 20; 1 km from Graskop to Sabie ploidy occurred in six of the nine species studied. (-D D ), Stirton 9798. 9865 A 9866. The different Rubus species varied in regard to their NATAL. — 2929 (Underberg): 25 km from Himeville to geographical distribution. Boesmansnek (-DC): Henderson A Gaum 50 A 51. 3029 (Kokstad): Ngeli Forest (-DA). Stirton 8135. a) The subgenus Eubatus R. iransvaliensis x R. longepedicellatus: The most widespread of the introduced Moriferi TRANSVAAL. — 2530 (Lydenburg): Nelspruit (-BD). Hen­ was Rubus affinis. Specimens representing R. affinis derson A Gaum 10. were collected in the northern Transvaal, Swaziland, Natal and southern and western Cape (Fig. I). How­ ever, the western Cape is the only place where this species invaded the natural vegetation and where TABLE 1.—Chromosome numbers of some South African Rubus this species is regarded as a weed. The somatic chro­ spccies mosome number of 28 for R. affinis (Gustafsson, 1933, 1939 & 1943; H eslop-Harrison, 1953) is con­ Species Plant No. 2n= firmed. Eubatus R. cuneifolius is restricted to Natal, with the ma­ Rubus affinis Stirton 5746 28 jority of specimens collected in western Natal (Fig. R. cuneifolius Stirton 8102 14 I). In addition to the chromosome number of 2n = Stirton 8154 14 Liengme s.n. 21 14 for R. cuneifolius reported by Shoem aker & Stur- Henderson <6 Gaum 93 28 rock (1959), polyploid forms with 21 and 28 somatic R. pascuus Henderson