Annals of Botany 85 : 91–101, 2000 Article No. anbo.1999.1000, available online at http:}}www.idealibrary.com on Aluminium Accumulation in Leaves of Rubiaceae : Systematic and Phylogenetic Implications S. JANSEN*†, S. DESSEIN†, F. PIESSCHAERT†, E. ROBBRECHT‡ and E. SMETS† †Laboratory of Plant Systematics, Botanical Institute, K.U.Leuen, Kard. Mercierlaan 92, B-3001 Heerlee, Belgium and ‡National Botanic Garden of Belgium, Domein an Bouchout, B-1860 Meise, Belgium Received : 12 July 1999 Returned for revision : 13 August 1999 Accepted : 21 September 1999 Aluminium (Al) accumulators are plants which accumulate more than 1000 ppm Al in their tissues. In addition to earlier analyses on leaves of Rubiaceae, 251 specimens were tested to verify the systematic importance of this character. The distribution of Al accumulators in the family shows that the feature is more or less restricted to a widely circumscribed subfamily Rubioideae including Craterispermeae, Knoxieae, Urophylleae and Pauridiantheae. Other Rubioideae representing strong Al accumulators are the genus Coccocypselum, Coussareeae, Prismatomerideae, and most Psychotrieae. It is hypothesized that the ability to accumulate high levels of Al evolved in an ancestor of the Rubioideae s.l. since the feature is concentrated in basal taxa of this subfamily. Moreover, recent phylogenetic insights in the subfamilial classification of the Rubiaceae based on macromolecular data are confirmed. Al accumulation is reported in only two genera outside Rubioideae s.l., viz. Coptosapelta and Alberta. Al accumulators generally are woody, relatively primitive taxa which occur in tropical forests with relatively high rainfall. In more derived Rubioideae such as Anthospermeae, Hedyotideae, Paederieae, Rubieae and Spermacoceae the tendency to herbaceousness is possibly associated with the absence of Al accumulation. It is likely that this lack is related to their adaptation to more xeric, alkaline soils and their more temperate distribution. # 2000 Annals of Botany Company Key words : Rubiaceae, aluminium accumulation, systematics, phylogenetic relationships, Rubioideae. INTRODUCTION Man’s perception of aluminium accumulation by plants is very old. In the posthumous Herbarium amboinense of Rumphius (1743) a tree was described as Arbor aluminosa or ‘ Aluyn-Boom ’ since the leaves and bark of this tree were used as a mordant instead of alum. Much of the early research on Al in plants was stimulated by the interest in plant materials as mordants for the traditional dyeing technology. Although most of these plants contained no colouring substances, the Al in their tissues served to set the colours generally furnished by other plants (Robinson and Edgington, 1945). Accumulation of minor elements in plants was defined by Robinson and Edgington (1945) as the uptake of a particular element ‘ in quantities very far above, sometimes many times above, the average quantity for normal plants ’. The mean content of Al in herbaceous tissues of plants is found to be 200 ppm or 0–02 % of the dry matter (Hutchinson, 1943). Hence, Chenery (1948 a) assumed Al accumulators to be plants that absorb Al in quantities more than 1000 ppm or 0–1 % of the dry matter in leaves ; plants with less Al in their leaves would be non- accumulating species. An exhaustive study of Al accumu- lation in herbarium samples from the British Colonies was made by Chenery (1948 a, b, 1949) ; Webb (1954) studied Al accumulators in the Australian-New Guinea flora, and Moomaw, Nakamura and Sherman (1959) contributed to * For correspondence. Fax : ›32–16–321968, email steven.jansen! bio.kuleuven.ac.be this topic by analysing Hawaiian plants. Examples of strong Al accumulators are Miconia (Melastomataceae), Symplocos (Symplocaceae), Vochysia (Vochysiaceae), or represen- tatives of Anisophylleaceae, Proteaceae, Rubiaceae and Theaceae (Metcalfe and Chalk, 1983). Numerous reports in the literature deal with the toxic effects of Al on non-tolerant plants (for a review see Lu $ ttge and Clarkson, 1992). Following Foy, Chaney and White (1978), plants that are tolerant to high Al concentrations can be divided into at least three groups according to the different mechanisms by which they avoid or diminish Al stress: (1) Al excluders which block the entry of Al at the root level ; (2) plants in which roots accumulate Al, preventing the element reaching the shoot ; and (3) plants with accumulation of Al in the tops. The last group includes plants defined as Al accumulators. From a systematic point of view, it is interesting to determine to what extent Al accumulators are related to taxonomic groups. Al accumulation clearly deserves little (or no) attention as regards its systematic importance at high taxonomic levels ; the character has arisen independently a number of times since it is scattered over more than 20 orders, including members of five of the six subclasses of the angiosperms (Cronquist, 1980). At a lower taxonomic level, Al accumu- lators are in only a few taxa suggested to have taxonomic value (e.g. Lycopodium : Hutchinson, 1943 ; Aniso- phylleaceae and Rhizophoraceae : Kukachka and Miller, 1980). Cronquist (1980) even states that ‘ the existence of accumulators and non-accumulators in the same family seems to be viewed with equanimity by all ’. 0305-7364}00}010091›10 $35.00}0 # 2000 Annals of Botany Company
Transcript
Annals of Botany 85 : 91–101, 2000Article No. anbo.1999.1000, available online at http:}}www.idealibrary.com on
Aluminium Accumulation in Leaves of Rubiaceae:
Systematic and Phylogenetic Implications
S. JANSEN*†, S. DESSEIN†, F. PIESSCHAERT†, E. ROBBRECHT‡ and E. SMETS†
†Laboratory of Plant Systematics, Botanical Institute, K.U.Leu�en, Kard. Mercierlaan 92, B-3001 He�erlee,
Belgium and ‡National Botanic Garden of Belgium, Domein �an Bouchout, B-1860 Meise, Belgium
Received: 12 July 1999 Returned for revision: 13 August 1999 Accepted: 21 September 1999
Aluminium (Al) accumulators are plants which accumulate more than 1000 ppm Al in their tissues. In addition toearlier analyses on leaves of Rubiaceae, 251 specimens were tested to verify the systematic importance of thischaracter. The distribution of Al accumulators in the family shows that the feature is more or less restricted to awidely circumscribed subfamily Rubioideae including Craterispermeae, Knoxieae, Urophylleae and Pauridiantheae.Other Rubioideae representing strong Al accumulators are the genus Coccocypselum, Coussareeae, Prismatomerideae,and most Psychotrieae. It is hypothesized that the ability to accumulate high levels of Al evolved in an ancestor ofthe Rubioideae s.l. since the feature is concentrated in basal taxa of this subfamily. Moreover, recent phylogeneticinsights in the subfamilial classification of the Rubiaceae based on macromolecular data are confirmed. Alaccumulation is reported in only two genera outside Rubioideae s.l., viz. Coptosapelta and Alberta. Al accumulatorsgenerally are woody, relatively primitive taxa which occur in tropical forests with relatively high rainfall. In morederived Rubioideae such as Anthospermeae, Hedyotideae, Paederieae, Rubieae and Spermacoceae the tendency toherbaceousness is possibly associated with the absence of Al accumulation. It is likely that this lack is related to theiradaptation to more xeric, alkaline soils and their more temperate distribution.
# 2000 Annals of Botany Company
Key words : Rubiaceae, aluminium accumulation, systematics, phylogenetic relationships, Rubioideae.
INTRODUCTION
Man’s perception of aluminium accumulation by plants isvery old. In the posthumous Herbarium amboinense ofRumphius (1743) a tree was described as Arbor aluminosa or‘ Aluyn-Boom’ since the leaves and bark of this tree wereused as a mordant instead of alum. Much of the earlyresearch on Al in plants was stimulated by the interest inplant materials as mordants for the traditional dyeingtechnology. Although most of these plants contained nocolouring substances, the Al in their tissues served to set thecolours generally furnished by other plants (Robinson andEdgington, 1945). Accumulation of minor elements inplants was defined by Robinson and Edgington (1945) asthe uptake of a particular element ‘ in quantities very farabove, sometimes many times above, the average quantityfor normal plants ’. The mean content of Al in herbaceoustissues of plants is found to be 200 ppm or 0±02% of the drymatter (Hutchinson, 1943). Hence, Chenery (1948a)assumed Al accumulators to be plants that absorb Al inquantities more than 1000 ppm or 0±1% of the dry matter inleaves ; plants with less Al in their leaves would be non-accumulating species. An exhaustive study of Al accumu-lation in herbarium samples from the British Colonies wasmade by Chenery (1948a, b, 1949) ; Webb (1954) studied Alaccumulators in the Australian-New Guinea flora, andMoomaw, Nakamura and Sherman (1959) contributed to
* For correspondence. Fax: 32–16–321968, email steven.jansen!bio.kuleuven.ac.be
this topic by analysing Hawaiian plants. Examples of strongAl accumulators areMiconia (Melastomataceae), Symplocos(Symplocaceae), Vochysia (Vochysiaceae), or represen-tatives of Anisophylleaceae, Proteaceae, Rubiaceae andTheaceae (Metcalfe and Chalk, 1983).
Numerous reports in the literature deal with the toxiceffects of Al on non-tolerant plants (for a review see Lu$ ttgeand Clarkson, 1992). Following Foy, Chaney and White(1978), plants that are tolerant to high Al concentrationscan be divided into at least three groups according to thedifferent mechanisms by which they avoid or diminish Alstress : (1) Al excluders which block the entry of Al at theroot level ; (2) plants in which roots accumulate Al,preventing the element reaching the shoot; and (3) plantswith accumulation of Al in the tops. The last group includesplants defined as Al accumulators. From a systematic pointof view, it is interesting to determine to what extent Alaccumulators are related to taxonomic groups.
Al accumulation clearly deserves little (or no) attention asregards its systematic importance at high taxonomic levels ;the character has arisen independently a number of timessince it is scattered over more than 20 orders, includingmembers of five of the six subclasses of the angiosperms(Cronquist, 1980). At a lower taxonomic level, Al accumu-lators are in only a few taxa suggested to have taxonomicvalue (e.g. Lycopodium : Hutchinson, 1943; Aniso-phylleaceae and Rhizophoraceae: Kukachka and Miller,1980). Cronquist (1980) even states that ‘ the existence ofaccumulators and non-accumulators in the same familyseems to be viewed with equanimity by all ’.
0305-7364}00}01009110 $35.00}0 # 2000 Annals of Botany Company
92 Jansen et al.—Al Accumulation in Rubiaceae
The Rubiaceae contain the largest number of Al accumu-lators of any family (Chenery, 1948b). Traditionally, thisfamily has been divided into two subfamilies : Cinchonoideaewith many ovules per ovary locule and Coffeoideae with oneovule per locule (Schumann, 1891). Bremekamp (1966;eight subfamilies) and Verdcourt (1958; three subfamilies)have introduced a series of new characters to delimitsubfamilies. Robbrecht (1988, 1994) used correlated evol-utionary trends to propose an emendation of theBremekamp}Verdcourt system, recognizing four sub-families : Cinchonoideae, Ixoroideae, Antirheoideae andRubioideae. Recent new insights in the family, however, areobtained from molecular data (e.g. Bremer and Thulin,1998; Andersson and Rova, 1999) and demonstrated thepolyphyletic nature of the subfamily Antirheoideae.
As part of a wood anatomical study of the Rubiaceae, wehave already investigated the occurrence of Al accumulationin wood of a wide array of representatives of the family(Jansen et al., unpubl. res.). The present paper includes adiscussion of Al accumulation in leaves since Al ispreferentially stored in these organs. The aim of the presentstudy is : (1) to overview the accumulation of Al in leaves ofrepresentatives of all tribes and subfamilies ; (2) to re-examine dubious results obtained by Chenery (1948a, b)and Webb (1954) ; and (3) to determine the taxonomic valueof the feature in view of recent phylogenetic insights into thefamily.
MATERIALS AND METHODS
Leaves of a selected number of Rubiaceae, representing allsubfamilies and tribes according to Robbrecht (1994), werecollected at the herbarium of the National Botanic Gardenof Belgium (BR). A single specimen of Amphidasya ambiguawas used from the herbarium collection of Utrecht (U).Since Al levels are found to be remarkably different betweenyoung leaves and mature ones (e.g. Chenery, 1955;Matsumoto et al., 1976; Cuenca and Herrera, 1988), onlymature leaves were used. All 251 specimens tested are listedin Table 1.
High Al concentrations in leaves can easily be detected bythe ‘aluminon’ test as described by Chenery (1946, 1948b).About 2 cm# of crushed leaf material is placed in a test-tube;2 ml of the ‘aluminon’ reagent (based on ammonium aurinetricarboxylate) is added and heated in a test tube at 100 °Cfor 5 to 10 min. If dust or soil remnants occur on leaves,they are washed by vigorous shaking in a 50% alcoholsolution in order to eliminate contamination (Chenery,1955). The ‘ aluminon’ test permits the assessment, byintensity of colour, of several levels of Al content rangingfrom about 300 to over 10000 ppm. Positive tests weredivided into three groups according to the colour of thereagent : a scarlet, crimson, or almost opaque colourindicates that the plant is a strong Al accumulator; a red todeep-red colour was interpreted as being characteristic ofintermediate accumulators ; and a pale red to pinkish redcolour demonstrates the accumulation of a relatively low Alcontent. A negative test is indicated by no change in thepinkish to orange colour of the reagent or only a darkerbrown colour.
RESULTS
All specimens tested are presented in alphabetical order inTable 1 according to their subfamilial and tribal position inRobbrecht’s (1994) classification of the Rubiaceae.
Cinchonoideae
Strong Al accumulators in the subfamily Cinchonoideaeare restricted to the tribes Urophylleae and Pauridiantheae,and the genera Coptosapelta (Coptosapelteae), Amphidasya,Gouldia, Raritebe and Temnopteryx (Isertieae). All thesespecies were found to be strongly positive (only Gouldia isintermediate) ; weak Al accumulators do not occur in theCinchonoideae.
Ixoroideae
All representatives of the Ixoroideae were negative in the‘aluminon’ test.
Antirheoideae
In the subfamily Antirheoideae several strong accumu-lators are found: Alberta (Alberteae), Craterispermum(Craterispermeae), and most specimens of Calanda andKnoxia (Knoxieae). Two specimens of Pentanisia (Knoxieae)are intermediate accumulators, and Canthium confertum(Vanguerieae) is only weakly positive. Other specimens ofthe Antirheoideae tested were negative.
Rubioideae
Most Al accumulators belong to the subfamilyRubioideae; all specimens of Coccocypselum (except thespecimen of C. guianense cultivated at the National BotanicGarden of Belgium), Coussareeae, Prismatomerideae, andnumerous Psychotrieae are strong Al accumulators. Otherstrong Al accumulators are Danais (Cinchoneae}Hedyotideae), Damnacanthus (group of Mitchella), Agathis-anthenum, Exallage (Hedyotideae), Saprosma (Paederieae)and Perama (Perameae). A few plants which accumulateless strongly are found in theHedyotideae,Argostemmateae,Schradereae, Psychotrieae, Morindeae, Paederieae, Antho-spermeae, Spermacoceae, and the genus Mitchella.
DISCUSSION
How �ariable is Al accumulation among Rubiaceae?
Our results indicate that aluminium accumulation is totallyabsent in numerous large taxa of Rubiaceae, such as theentire subfamily Ixoroideae and several tribes of theCinchonoideae and Antirheoideae sensu Robbrecht. On theother hand, Al accumulation is present in various species ofcertain genera and in many genera of particular tribes. Forthese taxa, Al tests are usually highly positive, indicatingthat these groups are strong Al accumulators. Thus thefeature is rather consistent for taxa in which strong Alaccumulators occur. Examples of strong accumulators arethe genera Colletoecema, Coptosapelta, Craterispermum,Lasianthus, Saprosma, and the tribes Coussareeae,
Jansen et al.—Al Accumulation in Rubiaceae 93
T 1. Al tests on lea�es of Rubiaceae
Subfamily – Tribe Specimens tested
CinchonoideaeCinchoneae Cinchona calisaya Wedd. (M. H. Weddell s.n.), C. pubescens Vahl (F. R. Fosberg & F. J. Hermann 21396) ;
Remijia ferruginea (A.St.-Hil.) DC. (col. unknown BR-S. P. 809973), R. ulei K.Krause (B. A. Krukoff 7228)Calycophylleae Alseis in�oluta K.Schum. (A. Glaziou 750) ; Calycophyllum spruceanum (Benth.) Hook.f. ex K.Schum. (J.
Isertieae Amphidasya ambigua Standley (P. J. M. Maas et al. 2732) ; Aoranthe cladantha (K.Schum.) Somers (J. J. F. E. deWilde et al. 32) ; Gonzalagunia brachyantha (A.Rich.) Urb. (C. Wright 233) ; Gouldia terminalis (Hook. & Arn.)Hillebr. (W. Robyns 2752) ; Heinsia crinita (Afzel.) G. Taylor (B. Sonke! 358) ; Mussaenda ferruginea K.Sch.(Clemens 948) ; Mycetia ja�anica (Blume) Reinw. ex Korth. (G. E. Edan4 o 7354 & 15356), M. longifolia (Wall.)Kuntze (Hooker & Thomson s.n. BR-S. P. 810209), M. nepalensis H.Hara (R. Stachey & J. E. Winterbottoms.n. BR-S. P. 809978) ; Raritebe palicoureoides Wernham (A. Tonduz 7977) ; Sabicea aspera Aubl.(Wullschlaegel 253), S. cinerea Aubl. (col. unknown BR-S. P. 810208), S. �illosa (J. Schunke-Vigo 11961; E.Martinez S. 19001) ; Schizostigma hirsutum Willd. ex Schult. (Thwaites 268) ; Temnopteryx sericea Hook.f. (F.Flewy 26463; J. J. F. E. de Wilde et al. 7)
Urophylleae Antherostele grandistipula (Merr.) Bremek. (A. Castro 5726) ; Praravinia everettii Merr. (G. E. Edan4 o 7211), P.mindanaensis (Elmer) Bremek. (C. A. Wenzel 2788) ; Urophyllum hirsutum (Wight) Hook.f. (A. D. E. Elmer21292)
Pauridiantheae Commitheca liebrechtsiana (De Wild. & T. Durand) Bremek. (J. Lebrun 799) ; Pauridiantha canthiifolia Hook.f.(F. J. Breteler 6514), P. floribunda (K.Schum. & K. Krause) Bremek. (B. Sonke! 1589) ; Rhipidantha chlorantha(K.Schum.) Bremek. (B. J. Harris & Mwasumbi 1148) ; Stelechantha cauliflora (Good) Bremek. (J. Lejoly86}303), S. ziamaeana (Jacq.-Fe! l.) N.Halle! (J.-G. Adam 27406)
Octotropideae Hypobathrum frutescens Bl. (G. E. Edan4 o 4032) ; Hyptianthera stricta (Roxb. ex Schult.) Wight & Arn. (G.Roxburgh s.n. BR-S. P. 809981) ; Lamprothamnus zanguebaricus Hiern (E. A. Mearns 2294; L. B. Mwasumbi10031) ; Scyphostachys coffeoides Thwaites (Thwaites 2710)
Korth. (A. D. E. Elmer 20614), C. oligocarpum Hiern (H. J. Schlieben 1379) ; (A. De Craene262) ; Pachystigma burttii Verd. (S. Bidgood et al. 566), P. latifolium Sond. (R. A. Dyer et al. 4708),P. pygmaeum (Schltr.) Robyns (S. Lisowski B-9670) ; Psydrax dicoccos Gaertn. (M. L. Merritt & F. W. Darling13915; col. unknown BR-S.P. 809984)
Guettardeae Chomelia martiana Mu$ ll.Arg. (col. unknown BR-S.P. 810212), C. spinosa Jacq. (E. Matuda 16404) ; Machaoniabrasiliensis (Hoffmanns. ex Humb.) Cham. & Schltdl. (B. Balansa 1767) ; Stenostomum coriaceum (Vahl.)Griseb. (Hahn 1440), S. granulatum Griseb. (C. Wright 1271)
Chiococceae Chiococca alba (L.) Hitchc. (P. Claussen 222)
Alberteae Alberta sp. (P. B. Phillipson et al. 4027), A. humblotii Drake (J. Randrianasolo & J. Marolhay 80), A. magnaE. Mey (R. G. Strey 5891; J. Gerstner 4850), A. minor Baill. (Service Forestier de Madagascar 7452), A.sambiranensis Homolle ex Cavaco (S. Malcomber et al. 1494) ; Nematostylis sp. (P. B. Phillipson et al. 3918)
Cephalantheae Cephalanthus glabratus (Spreng.) K.Sch. (A. M. R. Huidobro 4213)
Craterispermeae Craterispermum caudatum Hutch. (A. Jolly 305), C. cerinanthum Hiern (J. Louis 14717), C. schweinfurthii Hiern(D. Thomas 2795)
94 Jansen et al.—Al Accumulation in Rubiaceae
T 1. (Cont.)
Subfamily – Tribe Specimens tested
Knoxieae Calanda rubricaulis K.Schum. (P. Bamps et al. 4134) ; Knoxia manika (Verdc.) Puff & Robbr. (F. Malaisse & E.Robbrecht 2237), K. platycarpa Arn. (F. R. Fosberg & M.-H. Sachet 53337), K. sumatrensis (Retz.) DC. (N.Wallich 819) ; Paraknoxia par�iflora (Stapf. ex Verdc.) Verdc. (Polhill & Paulo 1986) ; Pentanisia prunelloides(Eckl. & Zeyh.) Walp. (O. Lincke 18), P. schweinfurthii Hiern (G. F. de Witte 06642), P. sykesii Hutch. (H. M.Richards 3913)
RubioideaeCinchoneae}Hedyotideae
Bou�ardia cordifolia DC. (G. B. Hinton 8433), B. longiflora (Cav.) Kunth (E. Ventura & E. Lo! pez 9585), B.tenuifolia Standl. (M. Martens s.n. BR-S.P. 809977) ; Danais fragrans (Lam.) Pers.(B. Randriamampionona334), D. volubilis Baker (G. Cremers 1618) ; Heterophyllaea fiebrigii (Krause) Standl. (J. R. De Sloover 346) ;Hindsia longiflora (Cham.) Benth. (P. Claussen 237A & 296A; col. unknown BR-S.P. 810204) ;Neohymenopogon parasiticus (Wall.) Bennet (N. Wallich 6113A); Oreopolus glacialis
(W. Lechler et al. 2895) ; Schismatoclada psychotrioides Baker (G. E. Schatz et al. 1376)
Hedyotideae Agathisanthenum globosum (Hochst. ex A.Rich.) Klotzsch (W. Robyns 2113) ; Arcytophyllum nitidum (Kunth)Schltdl. (J. Linden 409) ; Conostomium natalense (Hochst.) Bremek. (H. J. Schlieben 7146) ; Cruckshanksiamontiana Clos (M. C. Gay 133) ; Exallage rigida (Blume) Bremek. (Roxb.) Kurz. (M. Ramos & G. Edan4 o49808; A. D. E. Elmer 20691) ; Neanotis nummulariformis (Arn.) W. L. Lewis (F. R. Fosberg 58064),N. wightiana (Wall ex Wright & Arn.) W. H. Lewis (Griffith s.n. BR-S.P. 810218) ; Oldenlandia corymbosa L.(G. Schweinfurth 109), O. uniflora
..................L. (A. Glaziou 26) ; Oreopolus glacialis (Poep. & Endl.) Ricardi (T. M.
Pedersen 1654) ; Otomeria micrantha K.Schum. (J. Louis 7695) ; Pentas lanceolata (Forssk.) Deflers (Xing F.-W.et al. 85; (W. Robyns 2399), P. purpurea Oliv. (P. Bamps et al. 248), P. schimperiana (A.Rich.) Vatke (I. R.Dale 3266) ; Sacosperma paniculatum (Achten 140) ; Virectaria angustifolia (Hiern) Bremek. (J. Nemba & D. W.Thomas 321), V. major (K.Schum.) Verdc. (J. Louis 10971; P. Van der Veken 9390)
Ophiorrhizeae Ophiorrhiza acuminata DC. (G. E. Edan4 o 7406), O. glechomifolia Thwaites (Thwaites 1708), O. japonica Blume(M. Togashi 7003), O. nemorosa Thwaites (col. unknown BR-S.P. 802464) ; Spiradiclis bifida Kurz (N.Wallich 6216), S. caespitosa Blume (W. F. Winckel 1634B)
Coccocypseleae Coccocypselum sp. (Riedel 22), C. guianense (Aubl.) K.Schum. (Nat. Bot. Gard. Belgium, cultivation number83–27-84), C. guianense (Aubl.) K.Schum. (F. Billiet et al. 6238), C. tontanea Kunth (Riedel 182)
Schradereae Leucocodon reticulatum Gardner (Thwaites 347) ; Schradera sp. (C. A. Wenzel 3256), S. in�olucrata (Sw.)K.Schum. (Hahn 617), S. in�olucrata (Sw.) K.Schum. (R. A. Howard 16584)
Psychotrieae Amaracarpus sp. (M. M. J. v. Balgooy 669), A. sp. (P. van Royen & H. Sleumer 6204) ; Coryphotamnusauyantepuiensis (Steyerm.) Steyerm. (J. A. Steyermark 93934) ; Declieuxia cacuminis Mu$ ll.Arg. (W. R.Anderson et al. 35833), D. coerulea Gardner (J. H. Kirkbride et al. 1722), D. cordigera Mart. & Zucc. exSchult. & Schult.f. (H. S. Irwin et al. 8561), D. fruticosa (Willd. ex Roem. & Schult.) Kuntze (J. H. Kirkbride3736), D. fruticosa (Willd. ex Roem. & Schult.) Kuntze (G. Hatschbach 54074) ; Gaertnera bracteata E. M. A.Petit (J. Gillet 2685), G. paniculata Benth. (A. J. M. Leeuwenberg 8889), G. �aginans (DC.) Merr. (L. Pynaert37) ; Geophila afzelii Hiern (R. Letouzey 12597), G. cordifolia Miq. (Vasquez & Jaramillo 3500), G. macropoda(Ruiz. & Pav.) DC. (P. Jo$ rgensen 4081), G. repens (L.) J.R.Johnst. (Maas & Westra 3842) ; Hydnophytumformicarum Jack (V. Demoulin 5732) ; Margaritopsis nudiflora (Griseb.) K.Schum. (C. Wright 254) ; Metabolosdecipiens (Thwaites) Ridsdale (Thwaites 3093; F. R. Fosberg 58065) ; Pagamea guianensis Aubl. (M. RimachiY. 8254; F. Billiet & B. Jadin 1444; B. A. Krukoff 7083), P. plicata Spruce ex Benth. (L. Marcano-Berti & P.Salcedo 124–979) ; Palicourea crocea (Sw.) Schult. (col. unknown BR-S.P. 809988), P. officinalis Mart.(Glaziou 21531) ; Psathura borbonica J.F.Gmel. (Decaisne 6762) ; Psychotria caerulea Ruiz. & Pav. (Riedel2875), P. hoffmannseggiana (Wild. ex Roem. & Schult.) Mu$ ll.Arg. (W. H. A. Hekking 898), P. kirkii Hiern (A.Peter 52104), P. leiocarpa Cham. & Schltdl. (Riedel 631), P. pleiocephala Mu$ ll.Arg. (Martius 996), P.poeppigiana Mu$ ll.Arg. (H. von Tu$ rckheim 11252), P. variegata Steyerm. (B. A. Krukoff 7153), P. xantholobaMu$ ll.Arg. (A. F. Regnell 111–125) ; Rudgea coriacea (Spreng.) K.Schum. (Riedel 398), R. cornifolia (Humb. &Bonpl. ex Roem. & Schult.) Standl. (H. von Tu$ rckheim 1882)
? Psychotrieae Colletoecema dewevrei (De Wild.) E. M. A. Petit (J. Louis 8288; A. Sapin s.n. BR-S.P. 810207) ;Fergusonia zeylanica Hook.f. (Thwaites 284) ; Lasianthus cyanocarpus Jack (M. Ramos & G. Edan4 o 2–107) ;Litosanthes venulosus Deb & Garg (col. unknown in herb. Martius BR-S.P. 810191) ; Trichostachysmicrocarpa K.Schum. (R. Germain 2075, J. Gillet 810217)
Triainolepideae Triainolepis africana Hook.f. (S. Bidgood et al. 1497), T. africana Hook.f. (J. P. M. Brenan et al. 14512)
Morindeae Coelospermum crassifolium J. T.Johanss. (P. Bamps 5730), C. fragrans (Montrouz.) Baill. ex Guillaumin (H. S.MacKee 31146) ; Gynochthodes proboscidea Pierre ex Pit. (L. Pierre 4926) ; Morinda
...............sp. (W. Griffith s.n.),
M. citrifolia....................
L. (H. von Tu$ rckheim 6242), M. guatemalensis (Donn.Sm.) Steyerm. (Martinez 9238), M. rojoc L.(H. von Tu$ rckheim 2692A); Pogonolobus reticulatus F.Muell. (F. V. Mueller 6773)
Prismatomerideae Prismatomeris tetrandra (Roxb.) K.Schum. (C. A. Wenzel 2819), P. tetrandra (Roxb.) K.Schum. (W. Griffiths.n. BR-S.P. 809987) ; Rennellia morindiformis (Korth.) Ridl. (Dr. King 5432)
Jansen et al.—Al Accumulation in Rubiaceae 95
T 1. (Cont.)
Subfamily – Tribe Specimens tested
group of Mitchella Damnacanthus sp. (M. Martens s.n. BR-S.P. 810215), D. indicus P.Gaertn. (col. unknown BR-S.P. 802815) ;Mitchella repens L. (F. Cre!pin s.n. BR-S.P. 810195; F. Cre!pin 6746)
Coussareeae Coussarea nodosa (Benth.) Mu$ ll.Arg. (Riedel 264), C. rudgeoides Rusby (M. Rimachi Y. 8505) ; Farameaoccidentalis (L.) A.Rich. (R. Dechamps et R. Carrera 12139), F. multiflora A.Rich. (C.Presl.) Steyerm. (M.Rimachi Y. 1225)
Paederieae Kelloggia galioides Torr. (col. unknown BR-S. P. 810201) ; Paederia cruddasiana Prain (P. J. Greenway 8322),P. foetida.................
L. (M. D. Sulit 3624), P. pospischilii K.Schum. (Bally & A. R. Smith B14695) ; Putoria calabrica(L.f.) DC. (E. Cosson 3) ; Saprosma arboreum Blume (A. D. E. Elmer 21452), S. ternatum (Wall.) Hook.f.(N. Wallich 6248) ; Serissa japonica (Thunb.) Thunb. (Sino-American Guizhou Botanical Expedition 2252)
Anthospermeae Anthospermum herbaceum L.f. (W. Barbey 1427; J. Louis 4844), A. ternatum Hiern (F. Eyles 6183) ; Coprosmahirtella Labill. (Kaspiew 631), C. repens
..........A.Rich. (F. Von Mueller 6642) ; Leptostigma pilosum
...................................(Benth.)
Fosberg (E. Asplund 7171) ; Nertera granadensis..................................
(Mutis ex L.f.) Druce (C. G. Pringle 4672)
Spermacoceae Diodia teres Walter (G. V. Nash s.n. BR-S.P. 809970) ; Emmeorhiza umbellata (Spreng.) K.Schum. (Riedel 608;Riedel s.n. BR-S.P. 809967), E. umbellata
Sw. (A. Tandur 9144) ; Galianthe �alerianoides (Cham. & Schltdl.) Cabral (G. D. Lindsberg 108a) ; Richardiabrasiliensis Gomes (A. F. Regnell s.n. BR-S.P. 809975), R. grandiflora (Cham. & Schltdl.) Stend. (Pohl s.n.BR-S.P. 809976), R. scabra L. (F. R. Fosberg & N. Balakrishnan 53400) ; Spermacoce capitata Ruiz & Pav.(C. G. Pringle 2453), S. glabra
................Michx. (H. E. Ahles 57124), S. ocymoides Burm.f. (H. Pittier & T. Durand
7138), S. stachydea DC. (C. Geerling & J. Bokdam 1271), S. tenuior L. (C. G. Pringle 3254; H. Pittier 2871) ;Staelia scabra (C.Presl.) Standl. (C. G. Pringle 4477), S. �irgata (Link ex Roem. & Schult.) K.Schum. (P.Bamps 5172)
Tribus incertaePerameae Perama hirsuta Aubl. (G. Hatschbach 42432)inc. sed. Bothriospora corymbosa (Benth.) Hook.f. (S. McDaniel & M. Rimachi Y. 20527)
Specimens in bold are strong accumulators ; specimens underlined are intermediate accumulators ; specimens dotted underlined.............................
Pauridiantheae, Prismatomerideae, and Urophylleae. Inother taxa, however, Al accumulation is more variable sinceit is found in only few instances. The ‘aluminon’ tests inthese groups generally show moderate positive results.Accordingly, these taxa are regarded as intermediate orweak Al accumulators. Examples of moderate or weak Alaccumulators are specimens of Declieuxia, Geophila,Morinda and Schradera.
Furthermore, Al accumulation is not always consistentamong individuals of a species, since results vary whenspecimens are tested from different localities (see below).Hence, caution must be used in concluding, on the basis oflimited spot-testing, that a species is a strong accumulator,or even that accumulation does not occur under suitableconditions.
Comparison of our results with earlier analyses
Table 2 gives a summary of our results together withearlier Al tests of Rubiaceae. The data analysed in this studyare in good agreement with those presented by Chenery(1946, 1948a, b ) and Webb (1954). Core groups of Alaccumulators are Coussareeae, Coccocypselum, Prismato-merideae and Psychotrieae (Rubioideae sensu Robbrecht),as well as the Craterispermeae, Knoxieae (Antirheoideae),Urophylleae and Pauridiantheae (Cinchonoideae).
The following genera, which were shown to have at leastone positive specimen according to Chenery (1948a, b) orWebb (1954), were found to be negative in our tests :Cinchona and Remijia (Cinchoneae), Mycetia, Sabicea(Isertieae), Pa�etta (Pavetteae), Pachystigma (Vanguerieae),Lamprothamnus, Scyphostachys (Octotropideae), Saco-sperma (Hedyotideae), Psathura (Psychotrieae), Caelo-spermum (Morindeae), Richardia, Staelia (Spermacoceae),Galium and Rubia (Rubieae). Other genera were found to bepositive by us but negative in earlier tests : Coprosma,Leptostigma, Nertera (Anthospermeae), Diodia (Sperma-coceae), and Perama (Perameae). Except for Perama(Perameae), the latter genera are considered to be moderateor weak Al accumulators. Several factors may explain thesedifferences, such as interspecific variation or different en-vironmental conditions. Furthermore, it is likely that someof the specimens examined by Chenery (1948a, b) orWebb (1954) were wrongly identified herbarium material.Verification, unfortunately, is not possible because thename of the species tested is not mentioned and no referenceis made to herbarium material. Nevertheless, the number ofpositive specimens in several genera listed above issurprisingly low or even restricted to a single spot-test. Forinstance, the record of positive Al tests in the tribe Pavetteaeis most unusual, since Pa�etta would include only threepositive representatives from the 33 specimens analysed byChenery (1948b ; see Table 2). Moreover, more precise
Data according to Chenery (1946)", Chenery (1948a)#, Chenery (1948b)$, Webb (1954)%, and own tests& ; genera in bold have one or morepositive species, genera in italics are negative ; if known, the nominator between brackets gives the number of Al accumulating specimens, thedenominator is the total number of specimens tested
quantification of Al content is required for these taxa andcannot be obtained by the ‘aluminon’ test.
No material was available to test the Al content inMussaendopsis (Coptosapelteae), Acrobotrys (Rondeleti-eae), Indopolysolenia, Myrioneuron (Isertieae), Perakanthus(Vanguerieae) and Asemnantha (Chiococceae). Chenery(1948b) noted a positive test for these genera.
Systematic significance of Al accumulation in Rubiaceae
Cinchonoideae. In the Cinchonoideae sensu Robbrecht,the raphide-bearing tribes Urophylleae and Pauridiantheae
are characterized by strong Al accumulators. In recentcladograms based on molecular data, Pauridiantha andUrophyllum occupy a basal position within the subfamilyRubioideae (Bremer and Thulin, 1998; Andersson andRova, 1999). The presence of other strong Al accumulatorsin basal taxa of the Rubioideae (e.g. Coccocypselum,Colletoecema, Coussarea, Lasianthus) supports this position.
The distribution of Al accumulators in the Isertieae sensuRobbrecht supports the delimitation of the Isertieaeaccording to Bremer and Thulin (1998). Strong Al accumu-lators are Amphidasya, Gouldia, Temnopteryx, Raritebe andprobably also Indopolysolenia ; Mycetia and Myrioneuron
98 Jansen et al.—Al Accumulation in Rubiaceae
are suggested to be weak Al accumulators, while othergenera are non-accumulators. All Al accumulating generaare transferred to the Rubioideae (Fosberg, 1937;Andersson, 1996; Bremer and Thulin, 1998; Andersson andRova, 1999).
A remarkable Al accumulator is Coptosapelta (Copto-sapelteae), which has a problematic position since thesolitary, axillary, opposite flowers, T-shaped trichomes,and pororate pollen are aberrant in the tribe (Robbrecht,1994). The combination of raphides (Metcalfe and Chalk,1950; Fukuoka, 1980: Fig. 19B) and Al accumulationsuggests a position within the Rubioideae, although theplacentation of Coptosapelta seems to fit other Copto-sapelteae. It is clear that a revision and a sound morpho-logical and anatomical documentation of this genus isrequired.
Ixoroideae. Al accumulators do not occur in the subfamilyIxoroideae. As suggested above, the positive tests reportedby Chenery (1948b) for herbarium material of Pa�etta(Pavetteae), Lamprothamnus and Scyphostachys (Octo-tropideae) are probably due to misidentification of thematerial used, since specimens of these genera tested by usproved to be negative.
Antirheoideae. The subfamily Antirheoideae shows Alaccumulators in the Craterispermeae and the Knoxieae.Robbrecht (1988) stressed that raphides and heterostyly inthese two tribes are aberrant for the Antirheoideae. Ourobservations of Al accumulation in the Knoxieae confirmearlier associations of this tribe with Rubioideae. It wassuggested that the tribe Knoxieae be included in theRubioideae because of the presence of anthraquinones(Young et al., 1996). Bremer (1996) proposed mergingKnoxieae, Hedyotideae, and Spermacoceae into one largetribe—Spermacoceae s.l.—on the basis of rbcL sequencedata. The presence of raphides, heterostyly, and strongaccumulation of Al allows similar considerations for theposition of the monogeneric Craterispermeae in theRubioideae (Verdcourt, 1958).
More striking is the presence of Al accumulation in thegenus Alberta (Alberteae). This genus consists of six species,five of which are endemic to Madagascar. A. magna, whichis endemic to SE Africa, appears to be non-accumulating. Asingle specimen of the monotypic and closely related genusNematostylis tested by us proved to be negative. Puffet al. (1984) maintained the two genera in the Ixoroideaesensu Bremekamp, but stressed their isolated position andrather derived features. Robbrecht (1988), upon hisemendation of the Antirheoideae, included them in thatsubfamily on account of their solitary pendulous ovules.Absence of raphides, contorted corolla aestivation, andstylar pollen presentation contradict any association ofAlberta with Rubioideae. ITS and rbcL sequence datashow that Alberta should return to the Ixoroideae s.l.(Andreasen et al., 1999).
Rubioideae. This subfamily is characterized by numerousstrong Al accumulators ; Coussareeae, Prismatomerideaeand Psychotrieae are the tribes where Al accumulation ismost strongly expressed. In contrast with these taxa, themore herbaceous and}or relatively derived tribes of theRubioideae (Argostemmateae, Schradereae, Paederieae,
Anthospermeae, Spermacoceae and Rubieae) do not ac-cumulate Al or have few weak Al accumulators.Ophiorrhizeae, although occupying a basal position in theRubioideae, are entirely herbaceous and do not accumulateAl. Other non-accumulating taxa are the genusCruckshanksia, and the Hamelieae. The latter tribe hastraditionally been included in the Rubioideae because of thepresence of raphides (Verdcourt, 1958; Bremekamp, 1966;Robbrecht, 1994), but molecular data excluded this groupfrom the Rubioideae (Bremer et al., 1995; Manen andNatali, 1996).
The distribution of Al accumulators in the Rubioideaeconfirms several systematic affinities. The presence of strongAl accumulators in Coccocypselum and the Coussareeaecorroborates their close relationship as well as their ratherbasal position within the Rubioideae (Bremer, 1996; Bremerand Thulin, 1998; Andersson and Rova, 1999). However,the absence of a high Al level in leaves of Coccocypselumguianense growing in cultivation shows that the Al contentmay fall below the critical threshold. Coccocypselum is alsoclosely related with Declieuxia and Hindsia (Andersson andRova, 1999). While Declieuxia proved to accumulate Al inmost representatives tested, none of the specimens ofHindsia are positive.
Another example is that the weak Al accumulation inMorindeae s.str. supports the segregation of thePrismatomerideae, since the latter tribe includes all strongaccumulators (Igersheim and Robbrecht, 1994). Strong Alaccumulation also confirms the basal position within theRubioideae of Perama (Andersson and Rova, 1999), andDanais (Cinchoneae}Hedyotideae) (Bremekamp, 1952;Bremer, 1996). Furthermore, the absence of accumulationin the Triainolepideae contradicts its traditional associationwith the Psychotrieae. Manen and Natali (1996) postulatedthat Triainolepis probably does not belong to theRubioideae, but molecular data recently placed the genusnear the Knoxieae-Hedyotideae (Piesschaert et al., unpubl.res.), in which Al accumulators are restricted to few genera.
On the other hand, a close affinity between few taxa is notsupported by Al accumulation. The paleotropical genusGaertnera and its neotropical related genus Pagamea arerelated on the basis of several characters (Igersheim et al.,1994; Jansen et al., 1996), but they differ remarkably in theirAl uptake. Within the Paederieae, a tribe mainly consistingof non-accumulators or a few weak accumulators, the genusSaprosma is a remarkably strong Al accumulator. Thisgenus has traditionally been placed in Psychotrieae, but Puff(1992) transferred it to the Paederieae on the basis ofaestivation, pollen morphology, ovary and ovule structure,and the presence of paederoside. Note that this genus is theonly (large) tree of the Paederieae; other genera of this tribeare shrubs, or climbing shrubs.
Furthermore, the strong alliance between Mitchella andDamnacanthus (Robbrecht et al., 1991) is recently supportedby molecular data, but both genera are nested within theMorindeae (Andersson and Rova, 1999). While Damnacan-thus is a strong Al accumulator, the herbaceous Mitchellais a moderate accumulator. Al accumulation does notsupport a close relationship with the Morindeae s.str.,which contains no strong accumulators, but rather suggests
. Hypothetical summary of phylogenetic evidence from rbcL (Bremer and Thulin, 1998) and rps16 intron (Andersson and Rova, 1999) ;taxa in bold are strong AL accumulators.
a relationship with Prismatomerideae, Coussareeae, orprobably Psychotrieae (Manen and Natali, 1996; Nataliet al., 1996).
Phylogenetic and ecological implications of Alaccumulation in Rubiaceae
A plot of our data on a simplified tree representing recentphylogenetic insights based on rbcL (Bremer and Thulin,1998) and rps16 intron (Andersson and Rova, 1999)demonstrates that Al accumulation is mainly restricted toRubioideae and concentrated in the basal-most clades of thissubfamily (Fig. 1). It is most likely that the ability toaccumulate Al has evolved in a woody ancestor of theRubioideae and not in other subfamilies. The occurrence ofthe feature in Coptosapelta and Alberta suggests that thesystematic position of these genera requires further research.The distribution of Al accumulators supports the thesis thatthe character is predominantly confined to woody species(exceptions e.g. Coccocypselum).
In order to survive in most tropical rainforests, plantsmust either be able to tolerate the presence of Al in theirtissues or they must have developed some mechanism forexcluding it. It has been suggested on the basis of statisticalcorrelations by Chenery and Sporne (1976) that primitivedicotyledons are characterized by the former alternative. Asregards Rubiaceae, the Rubioideae are generally acceptedto be the most derived subfamily, showing the most extremeevolution to herbaceousness and adaptation to temperateregions and xeric areas in the Rubieae (Robbrecht, 1988).Within the Rubioideae, however, Al accumulation can beconsidered to be a rather primitive feature. Wood hasevolved secondarily in some taxa of the Rubioideae (e.g.Crucianella, Galium, Relbunium, Rubia : Koek-Noorman,1977; Rubia : Carlquist, 1992), but these groups do notappear to be Al accumulators.
We suppose that Al accumulation is at least partly due togenetic control of metabolic pathways in Rubiaceae, since
its occurrence is generally restricted to related groups.Cuenca et al. (1990, 1991) suggested that the main differencebetween Al accumulators and non-accumulators is thedifference in permeability of the endodermic cells to Al$+.The subfamily Rubioideae clearly differs from otherrubiaceous groups not only in Al uptake, but also inother characters that are probably related to specificmetabolic pathways. The presence of raphides, for instance,has long been considered to characterize all groupsassociated with Rubioideae and present insights confirm thetaxonomic value of this character. Another interestingchemosystematic marker in the Rubioideae is the presenceof anthraquinones (Young et al., 1996). Also, the subfamilyis characterized by a deletion of one of the atpB promotorsin the cpDNA (Manen and Natali, 1996).
Al accumulation, however, is also partly determined byenvironmental (edaphic) conditions (e.g. available supply ofsoluble Al in the soil, soil pH). This is illustrated, forinstance, by the absence of Al accumulation in Cocco-cypselum guianense cultivated in a greenhouse of theNational Botanic Garden of Belgium, but all tests appliedon herbarium material of Coccocypselum growing in itsnatural habitat proved to be strongly positive. Anotherexample is that Al accumulation does not occur in epiphytes,for instance some species of Ophiorrhiza (Ophiorrhizeae),Schradera (Schradereae), Myrmecodia, Hydnophytum(Psychotrieae) and Coprosma (Anthospermeae).
Al accumulators generally are confined to leached acidsoils in relatively high rainfall areas (cloud forests) ; drierareas with alkaline soils or forests with relatively lowrainfall have no Al accumulators (Webb, 1954; Lu$ ttge,1997). There is usually a close correlation between soil pHand the concentration of soluble Al; but this correlationdoes not always occur (Harris, 1961).
All Al accumulators found in Rubiaceae are from tropicalregions, but we have no information on their preciseenvironmental conditions since these data are frequently
100 Jansen et al.—Al Accumulation in Rubiaceae
poor on herbarium labels, and complete ecological obser-vations of accumulators are beyond the scope of the presentstudy. Also, a more precise method for quantification of Alcontent is required for the consideration of environmentalinfluences. Nevertheless, it is hypothesized that rubiaceousAl accumulators are tropical and mostly woody taxa. Theabsence (or loss) of Al accumulation in the Rubioideae issupposed to be associated with adaptation to xeric con-ditions with much less rainfall and less acid soils. Hence, thispossibly represents the evolution of the more herbaceousRubioideae to temperate regions. Similarly, Webb (1954)suggested that the inability of accumulation has accom-panied the adaptation of certain groups (e.g. Proteaceae) toxeric conditions and less acid soils. Moreover, changes inthe regulating elements of the atpB promotors in Rubioideaeare postulated to have evolutionary implications in thecourse of adaptation to the herbaceous habit and in thecolonization of temperate and xeric areas (Manen andNatali, 1996).
Relationship between Al accumulation and colour of leaf,fruit and flower
Hallier (1922) and Chenery (1948a, b) concluded that Alaccumulators have thick leaves and a characteristic yellow-green colour in herbarium material. Hutchinson (1943) andHutchinson and Wollack (1943), however, suggested thatthis relationship is weak since the criteria are not universal.While collecting leaves from herbarium material ofRubiaceae, yellow-green leaves were frequently found inAl accumulating tribes such as the Craterispermeae,Pauridiantheae, Prismatomerideae and Urophylleae.Although the correlation is not always high and theappearance of the dry leaf cannot replace the ‘aluminon’test, leaf colour, in some instances, permits an easy andquick method of identifying potential Al accumulators.Nevertheless, the colour of leaves from herbarium materialis also dependent on the method of drying, age, chemicaltreatment etc. Note that Al is mainly stored in the palisadeparenchyma cell walls (Cuenca et al., 1991; Haridasanet al., 1986).
Chenery (1946, 1948a, b) has also noted that the colour offruit and flowers in Al accumulators is frequently blue. Thiscorrelation is, to a certain degree, confirmed in Rubiaceae(e.g. Coussareeae). On the other hand, not all blue-floweredor -fruited plants are Al accumulators.
Ni hyperaccumulation in Rubiaceae
Ni hyperaccumulation in Rubiaceae was first noted in theNewCaledonian speciesPsychotria douarrei (G. Beauvisage)Da$ niker (Jaffre! and Schmid, 1974). This species is the onlyNi hyperaccumulator among 210 Psychotria species studiedfrom the Pacific Basin (Baker et al., 1985). Hyperaccumu-lation of Ni was also found by Reeves et al. (1999) inCuban serpentine species of Ariadne, Phyllomelia (generaincertae sedis), Rondeletia (Rondeletieae), and in at leastfive Cuban species of Psychotria. Although studies of Nihyperaccumulation are more limited, its taxonomic impli-cations in Rubiaceae appear to be significant at the specificor generic level. Genera such as Psychotria, including both
hyperaccumulators and non-accumulators of Ni, may beinteresting taxa for classification purposes among species.The most unifying feature of taxa which hyperaccumulatethis metallic element is their restricted distribution and highdegree of endemism (e.g. Baker and Brooks, 1989).
CONCLUSIONS
Analyses of the Al content in rubiaceous leaves demon-strated the usefulness of Al accumulation as a chemo-taxonomic marker for a widely circumscribed subfamily‘Rubioideae’ including Craterispermeae, Knoxieae,Pauridiantheae, and Urophylleae. Within the Rubioideae,the more herbaceous and derived taxa show a retention ofthe character since these tribes have only few weak Alaccumulators. The lack of Al accumulation in these taxa isprobably correlated with the adaptation to more xeric,alkaline soils and is associated with a more temperatedistribution. The occurrence of the character in Coptosapelta(Coptosapelteae–Cinchonoideae) and Alberta (Alberteae–Ixoroideae) is more difficult to explain at present ; thetaxonomic position of both genera needs further research.All other Cinchonoideae and Ixoroideae are non-accumu-lators. It is concluded that Al accumulation is a chemo-taxonomic character demonstrating relationships between anumber of taxa at the base of the Rubioideae. In addition toother features, the Al test can also be useful for auxiliaryidentification purposes at the generic level.
ACKNOWLEDGEMENTS
We thank Anja Vandeperre for assisting with the ‘aluminon’tests. Steven Jansen holds a scholarship of the ResearchCouncil of the K.U.Leuven. This research is supported bya grant from the Research Council of the K.U.Leuven(OT}97}23) and by grants from the Fund for ScientificResearch–Flanders (F. W. O., Belgium): project numberG. 0143±95.
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