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IAWA Journal, Vol. 21 (2), 2000: 197–212

ALUMINIUM ACCUMULATION IN :

AN ADDITIONAL CHARACTER FOR THE DELIMITATION OF THE

SUBFAMILY ? by S. Jansen1, E. Robbrecht2, H. Beeckman3 & E. Smets1

SUMMARY

The chrome azurol-S test, which is a chemical spot-test for Al accumu- lation in wood, was applied to 443 wood samples of members of the Rubiaceae. A positive reaction was found in 103 specimens. Compari- son of the results with earlier analyses of of Rubiaceae shows that Al accumulation occurs more frequently in leaves than in wood. The strongest Al accumulators occur in the neotropical genera Psy- chotria subg. Heteropsychotria, , , and . The distribution of Al accumulators is discussed in view of recent tribal and subfamilial classification of the Rubiaceae. The major conclusion is that Al accumulation is almost limited to the subfamily Rubioideae. Within the Rubioideae, however, not all tribes show the character, es- pecially the predominantly herbaceous , Paederieae, , and . Al accumulation in the Urophylleae, Pauri- diantheae, Craterispermeae, and supports earlier associations of these tribes with the Rubioideae. Key words: Aluminium accumulation, chrome azurol-S test, chemo- , Rubiaceae, Rubioideae.

INTRODUCTION

Plants containing a high level of Al in their above-ground tissues (more than 1,000 ppm / dry weight) are termed ʻaluminium plantsʼ or ʻaluminium accumulatorsʼ (Hutch- inson & Wollack 1943; Hutchinson 1945; Robinson & Edgington 1945). They have mainly been recorded by Chenery (1946, 1948a, b, 1949), Webb (1954), and Moomaw et al. (1959). In these studies, the high Al content is detected by the ʻaluminonʼ test (based on ammonium aurine tricarboxylate) applied to leaves of living or dried speci- mens. At present, the number of known accumulating families has increased to about 45. Al accumulators are especially common in families such as Anisophylleaceae, Celastraceae, Cornaceae, Diapensiaceae, Geissolomataceae, Grossulariaceae, Melas-

1) Laboratory of Systematics, Institute of and Microbiology, K.U. Leuven, Kard. Mercierlaan 92, B-3001 Leuven, Belgium. 2) National Botanic Garden of Belgium, Domein van Bouchout, B-1860 Meise, Belgium. 3) Royal Museum for Central , Leuvensesteenweg 13, B-3080 Tervuren, Belgium.

Downloaded from Brill.com09/24/2021 04:57:17PM via free access 198 IAWA Journal, Vol. 21 (2), 2000 tomataceae, Pentaphylacaceae, Polygalaceae, Proteaceae, Rubiaceae, Symplocaceae, Theaceae, and Vochysiaceae (Chenery & Sporne 1976; Metcalfe & Chalk 1983). They are in general woody inhabiting tropical or subtropical regions. Above the level, Al accumulation has been accorded very little taxonomic significance. The fami- lies listed above belong to different major groups of the dicotyledons, and it is beyond doubt that the character has arisen a number of times in plant evolution. A chemical spot-test for Al and its application for wood identification was devised by Kukachka and Miller (1980). Although these investigators used a different stain (a chrome azurol-S solution) from that employed by Chenery and others, many of their results confirm earlier findings. Almost all families which showed a positive chrome azurol-S test were represented in the list of families given in Chenery and Sporne (1976) and Metcalfe and Chalk (1983). Apart from a small number of more recent papers (e.g., Quirk 1980: Vochysiaceae; Bridgwater & Baas 1982: Xanthophyl- lum; Keating & Randrianasolo 1988: Anisophylleaceae) which briefly refer to some of the results published by Kukachka and Miller (1980), very few new studies on chrome azurol-S tests are reported in literature, despite the fact that the spot-test is included in the IAWA list of standard wood characters as feature 216 (IAWA Com- mittee 1989). Since Chenery (1948b) stated that the Rubiaceae contain the largest number of Al accumulators of any family, with 647 in 91 genera, the present study aimed to determine the taxonomic significance of Al accumulation in this very large family. Chrome azurol-S tests were applied to 443 wood samples representing all subfamilies and tribes of the family (except for some small herbaceous tribes such as and Argostemmateae). In addition, the results on Al accumulation in rubiaceous taxa obtained by Chenery (1946, 1948a, b) and Webb (1954) are summarised. Thus, it is possible to compare accumulation in wood and leaves, and evaluate the information in the light of recent systematic insights. The intrafamilial classification of the - ceae has drastically changed since the publication of these early works, and still is in a state of flux (e.g., Robbrecht et al. 1996; Bremer & Thulin 1998; Andersson & Rova 1999). The discussion of our results follows Robbrechtʼs (1994) classification of the family, with reference to recently proposed modifications.

MATERIALS AND METHODS

The material investigated came from the xylaria of Madison (MADw-SJRw), Mont- pellier (CTFw), Tervuren (Tw), and Utrecht (Uw), from the herbaria of Brussels (BR), Kew (K), Leiden (L), Missouri (MO), Paris (P), and Wageningen (WAG), and from living collections in the greenhouses of the National Botanic Garden of Belgium. The number of specimens that show a positive Al test/total number of specimens tested is given in brackets; e.g. Coussarea (11/11) means that all 11 specimens of Coussarea tested gave a positive Al test. The presence of high Al concentrations in wood was detected by use of a 0.5% solution of chrome azurol-S as described by Kukachka and Miller (1980). This solu- tion has a yellow to orange colour. One or two drops of the solution are applied to the

Downloaded from Brill.com09/24/2021 04:57:17PM via free access Jansen, Robbrecht, Beeckman & Smets — Al accumulation in Rubiaceae 199 freshly exposed end-grain surface of the wood sample, because dirt and other con- taminants may affect the test. Also, we applied the solution to the transverse side of the wood block, since uptake readily occurs in this direction. Wood samples produc- ing a blue to dark blue colour indicate a positive reaction and are regarded as strong accumulators. A light purple to bluish colour shows that the test is intermediate which is characteristic for weaker Al accumulators. Wood that contains a low Al concentra- tion does not change colour in the presence of chrome azurol-S solution; these speci- mens are considered to be negative. The Al test was repeated in the case of dubious and intermediate reactions. The chrome azurol-S solution can easily be used to test woody parts of specimens by making a fresh cut a few mm2 into the sur- face before applying the test solution.

RESULTS

Results of the chrome azurol-S tests are given in column I of Table 1. In 89 specimens the wood produced a (bright) blue colour in a matter of minutes. In 14 specimens the chrome azurol-S test gave an intermediate reaction. Most of these woods required five to ten minutes before a change of colour was visible. Within the sensu Robbrecht, positive specimens occurred in the tribes Pauridiantheae (, Poecilocalyx, and Stelecantha) and Urophylleae (Leucolophus, Maschalocorymbus, , and ). No accumulators were found in the subfamily . In the Antirheoideae sensu Robbrecht, posi- tive taxa were restricted to the monogeneric Craterispermeae, and some genera of the Knoxieae, namely Calanda, and . As for the subfamily Rubi- oideae, many wood samples reacted positively. We observed high frequencies for accumulation in the neotropical genera subg. Heteropsychotria (formerly placed in Cephaelis) (5/6), Coussarea (11/11), Faramea (13/13), and Rudgea (9/10). Other Rubioideae proved to be marked accumulators, viz. (3/4), (3/3), (7/9), (4/5), (3/3), (2/2), Tri- chostachys (2/2), and the single specimen tested of (1/1). In contrast, only few representatives of (2/6), (1/4), (1/6), (3/10), and Psychotria p.p. reacted positively. The secondary xylem of Lasianthus acuminatus, Saprosma ceylanica, and S. ternatum was found to be nega- tive, but the outermost part near the bark gave a positive reaction. For most species investigated here, only one wood sample was tested. However, from 40 species two or more specimens were investigated. The specimens were all negative in 26 species; the two wood samples of 5 species (Faramea anisocalyx, Pentanisia renifolia, Prismatomeris beccariana, Psychotria cotejensis, Trichostachys microcarpa) proved to be positive. Positive and intermediate reactions were found in 4 species ( cerinanthum, Faramea occidentalis, Lasianthus batangen- sis, Psychotria vasiviensis). In contrast, the wood samples of Colletoecema dewevrei, Maschalocorymbus corymbosus, micrantha, Pentanisia schweinfurthii, and Psychotria peduncularis differed in their reaction; the tests gave negative as well as positive or intermediate results for these species.

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Table 1. Summary of tests on Al accumulation in wood or leaves of Rubiaceae; genera in bold have one or more positive species; genera underlined are intermediate; genera in italic are negative; if known, the nominator between brackets gives the number of Al accumulating specimens, the denominator is the total number of specimens tested. Chrome azurol-S tests in column II are after Kukachka & Miller (1980)5 and Rogers (1981, 1984)6; data in column III from Chenery (1946)1, Chenery (1948a)2, Chenery (1948b)3, Webb (1954)4. Classification following Robbrecht (1994).

Subfamily / Tribe I: Own chrome II: Chrome azurol-S III: Al tests on leaves azurol-S tests tests from literature

CINCHONOIDEAE (0/1); Ferdinandusa5 Cinchona (1/4)3; (0/5); Ferdinandusa3; (0/1); Remijia (1/5)3; 3 other genera negative 6 other genera negative Calycophylleae 2 genera negative 4 genera negative Coptosapelteae (0/1); Coptosapelta (7/7)3; Mussaendopsis (0/1); Mussaendopsis (1/2)3; 5 other genera negative 6 other genera negative 5 genera negative Hillieae 3 genera negative 2 genera negative Henriquezieae (0/1); Henriquezia5, 6; Henriquezia3; Platycarpum3 (0/1); Gleasonia6 (3/3); (0/1) Platycarpum6 (1/4) Rondeletieae 7 genera negative (1/1)3; 9 other genera negative Simireae Simira3 Sipaneeae Sipanea3 5 genera negative group , Exostemma 9 genera negative Isertieae (0/1); Amphidasya (1/1)3; (0/2); Indopolysolenia (1/1)3; Temnopteryx (0/1); Mycetia (1/1)3; 5 other genera negative Myrioneuron (2/4)3; Sabicea (1/1)3; Temnopteryx (1/1)3; 7 other genera negative Urophylleae Leucolophus (1/1); Urophyllum5 Pleiocarpidia (4/4)3; Maschalocorymbus (2/3); Praravinia (23/23)3; Pleiocarpidia (0/3); Urophyllum (41/41)3 Praravinia (5/5); Urophyllum (3/4) Pauridiantheae Commitheca (0/1); Pauridiantha5 Commitheca (1/1)3; Pauridiantha (4/6); Pauridiantha (5/5)3; Poecilocalyx (1/1); Poecilocalyx (1/1)3 Stelecantha (1/1) →

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Subfamily / Tribe I: Own chrome II: Chrome azurol-S III: Al tests on leaves azurol-S tests tests from literature

IXOROIDEAE 30 genera negative 29 genera negative Pavetteae (0/3); Pavetta (3/37)3; 13 other genera negative 6 other genera negative 2 genera negative 2 genera negative Aulacocalyceae 2 genera negative 3 genera negative Octotropideae Lamprothamnus (0/1); Lamprothamnus (1/1)3; 4 other genera negative Scyphostachys (1/1)3; 12 other genera negative ANTIRHEOIDEAE Retiniphylleae Retiniphyllum (0/1); Canthium (14/34)3, 4; (0/1); Pachystigma (1/4)3; 4 other genera negative Perakanthus (1/2)3; 6 other genera negative 5 genera negative 11 genera negative Chiococceae 3 genera negative Asemnantha (1/1)3; 6 other genera negative Alberteae Alberta (0/2) Alberta (2/2)3; Nematostylis Cephalantheae Cephalanthus Craterispermeae Craterispermum (4/4) Craterispermum5 Craterispermum (10/10)2, 3 Knoxieae Calanda (1/1); Calanda (1/1)3; Chlorochorion (0/2); Knoxia (1/3)3, 4; Knoxia (1/2); Pentanisia (11/13)2, 3 Pentanisia (3/4)

RUBIOIDEAE Cinchoneae / Danais (3/4) Danais (17/18)3; Hedyotideae (2/5)2,3; 4 other genera negative Hedyotideae (1/2); Gouldia5 (probably Hedyotis (11/14)3, 4; Hedyotis (3/3); Hedyotis) (5/13)3, 4; Oldenlandia (0/1); Otomeria (3/9)3; Otomeria (1/6); Phyllocrater (1/1)3; Sacosperma (0/1); Sacosperma (3/3)2, 3; 6 other genera negative (1/3)3, 4; 17 other genera negative Ophiorrhizeae Ophiorrhiza3, Spiradiclis3 Coccocypseleae (herbaceous) (27/27)1, 2, 3 Argostemmateae (herbaceous) Argostemma3, Neurocalyx3 Hamelieae 4 genera negative →

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Subfamily / Tribe I: Own chrome II: Chrome azurol-S III: Al tests on leaves azurol-S tests tests from literature (Rubioideae ctd) Schradereae (0/3) Schradera (3/7)3; Lecananthus3, Leucocodon3 (0/2); Psychotria5 (8/11)2, 3; (0/1); (as Calycodendron); Calycosia (3/3)3; Gaertnera (1/4); Calycosia5; Cephaelis (62/72)1, 2, 3; Margaritopsis (1/1); Cephaelis5; Chassalia (7/10)1, 2, 3 Palicourea (3/10); Gillespiea5; Coelopyrena (1/1)3; Psychotria (11/36); Palicourea5; Declieuxia (12/13)1, 2, 3; Rudgea (9/10); Psychotria p.p.5; Gaertnera (6/6)2; 4 other genera negative Rudgea5 (2/8)2, 3, 4; Hedstromia (1/1)3; (3/18)3, 4; Margaritopsis (1/1)3; Metabolos (1/1)3; Palicourea (69/70)1, 2, 3; Psathura (3/3)3; Psychotria (207/366)1, 2, 3, 4; Rudgea (22/22)1, 2, 3; (1/1)3; Stachyococcus (2/2)2, 3; 3 other genera negative ?Psychotrieae Colletoecema (2/6); Fergusonia (1/1)3; Lasianthus (7/9); Lasianthus (61/62)1, 2, 3, 4; Trichostachys (2/2) Trichostachys (1/1)3 Triainolepideae Triainolepis (0/3) Triainolepis (0/1)3 Morindeae (0/3); Appunia (4/5)3; Morinda (0/12) Caelospermum (2/3)1, 3, 4; Morinda (8/22)3, 4 Prismatomerideae Prismatomeris (4/5); Prismatomeris (10/10)3; Rennellia (3/3) Rennellia (3/3)3 group of (0/1) Damnacanthus (1/1)3; (Mitchella is herbaceous) Mitchella (1/1)3 Coussareeae Coussarea (11/11); Coussarea5; Coussarea (8/8)1, 3; Faramea (13/13) Faramea5 Faramea (83/83)1, 2, 3 Paederieae Saprosma (2/2); Saprosma (15/15)1, 2, 3; 7 other genera negative 10 other genera negative Anthospermeae (0/5); Coprosma (1/11)2, 3, 4; 6 other genera negative 10 other genera negative Spermacoceae (0/1); (1/1)3; Emmeorhiza (0/1); (2/9)3, 4; (0/4); Spermacoce (10/18)1, 2, 3, 4; (0/1) Staelia (1/1)3; 6 other genera negative Rubieae (0/1); (2/12)3; Rubia (1/4)3; Rubia (0/1) 8 other genera negative inc. sed. 3 genera negative 16 genera negative

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DISCUSSION

Comparison of column I with column II of Table 1 There is a good general agreement between our observations and earlier results. Kukachka and Miller (1980) and Rogers (1981, 1984) detected the major rubiaceous groups in which Al accumulation is dominant, namely the Psychotrieae and Cous- sareeae, and the Urophylleae–Pauridiantheae complex, as well as the genera Hedyotis and Craterispermum. We were unable to obtain any material to study the genera Calycosia and , which, according to Kukachka and Miller (1980), are both Al accumulators. We have encountered only four dubious records in this earlier data set, viz. Ferdi- nandusa (Cinchoneae), and three genera of the Henriquezieae (Gleasonia, Henriquezia, and Platycarpum). Contrary to the intermediate reactions recorded for Ferdinandusa cf. paraensis (Kukachka & Miller 1980), and Gleasonia and Platycarpum (Rogers 1981, 1984), all five species ofFerdinandusa and a single specimen of Gleasonia and Platycarpum tested by us gave a negative reaction. While Kukachka and Miller (1980) reported at least one positive species of Henriquezia, a herbarium specimen of Henriquezia verticellata proved to be negative; no mature wood sample was avail- able. Furthermore, it is interesting to note that Al does not accumulate in leaves of Ferdinandusa, Henriquezia, and Platycarpum (Chenery 1948b).

Comparison of column I and II with column III of Table 1 There is also good general agreement between wood data (earlier and present) and published documentation on accumulation of Al in rubiaceous leaves. The total number of genera that have at least one specimen with a positive chrome azurol-S test is found to be 29 (103 specimens); the total number of genera accumulating Al in their leaves, however, is 76 (830 specimens). Thus, in quite a number of instances, ac- cumulation is recorded for genera with no accumulation in the wood: e.g., Coptosapel- ta (Coptosapelteae), Pleiocarpidia (Urophylleae), Canthium (Vanguerieae), Alberta (Alberteae), Chassalia and Declieuxia (Psychotrieae), Morinda (Morindeae), and Spermacoce (Spermacoceae). The reverse situation, Al accumulation in wood but not in leaves is not found, except for the dubious records discussed in the preceding para- graph. Although for several genera mentioned in column III no wood samples were available, it is clear that Al accumulates preferentially and more strongly in leaves than in wood. Since Kukachka and Miller (1980) found that all the wood samples showing a positive chrome azurol-S test had an Al content of 1,000 ppm or more in their wood, a different level of detection between the ʻaluminonʼ test and the chrome azurol-S test seems unlikely. The cell wall of palisade parenchyma cell walls is the main Al sink in Al accumulators as demonstrated for instance by Cuenca et al. (1991). It is possible that bounding of Al to the cell wall of leaves is a mechanism whereby the element can be detoxified by storage and removed from the plant by leaf fall. Similarly, Ernst (1972) suggested that leaf fall could lead to removal of Ni in Indigofera setiflora Bak., whereas in perennial organs such as roots the same mecha- nism could not be operative.

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Furthermore, the wood samples of Saprosma and Lasianthus acuminatus suggest that the amount of Al is much higher in the outermost part of the secondary xylem, since only wood near the bark was found to be positive for the chrome azurol-S test. Also, it is interesting to note that the wood samples of both stem and root of Pentanisia renifolia tested are positive. An important point to stress is that not all genera giving a positive score in column I, II, or III do accumulate the same quantities of Al. Amongst members of the Rubiaceae the strongest accumulators found by Chenery (1946, 1948a) were Psychotria muscosa (as ʻCephaelis muscosaʼ, 13,700 ppm), Faramea anisocalyx (36,900 ppm), F. eury- carpa (35,000 ppm), F. insignis (40,000 ppm), Palicourea nigricans (17,100 ppm), Psychotria brachiata (15,400 ppm), P. herzogii (22,600 ppm), and Rudgea justicoides (36,800 ppm). These marked accumulators have an Al content higher than 10,000 ppm and belong to the Psychotrieae or Coussareeae. Unfortunately, very little or no quantitative data are given by Chenery (1946, 1948a, b) and Webb (1954) to illus- trate the range of Al concentration in the plants tested. We suggest that the number of positive representatives within a or a tribe is an indication of the relative Al concentration in that group. It is clear that comparison of Al accumulation in leaves and wood deserves quantification.

The taxonomic value of Al accumulation in Rubiaceae The chrome azurol-S test is generally thought to be consistent at the generic level ( Kukachka & Miller 1980). They stated that in larger complex genera such as Psych- otria (Psychotrieae, Rubiaceae), and Miconia (Melastomataceae) all species do not test positive. With regard to Psychotria, one can answer that the polyphyletic nature of this genus is well known (Andersson & Rova 1999). Our results indicate that a posi- tive reaction is generally consistent in the genera Coussarea (11/11), Craterispermum (4/4), Faramea (13/13), Lasianthus (7/9), Praravinia (5/5), Prismatomeris (4/5), Psychotria subg. Heteropsychotria (5/6), Rudgea (9/10), Trichostachys (2/2), and Urophyllum (3/4), but more variable in genera as for instance Colletoecema (2/6), Gaertnera (1/4), and Palicourea (3/10). Therefore, the spot-test is for some genera useful for wood identification purposes. Indeed, a specimen assigned to Faramea eurycarpa was found to be the only Faramea material that reacted negatively; light microscopic observation of wood sections of this specimen confirmed that it had been wrongly identified. In the Cinchonoideae, Al accumulation is restricted to few tribes. In the Isertieae sensu Robbrecht, some genera show leaf but no wood accumulation. Interestingly, the distribution of the leaf accumulation supports Bremer and Thulinʼs (1998) delimi- tation of the Isertieae. No accumulation has been observed in (Isertieae) and and (), while all Al accumulating genera (except Sabicea) are transferred to the Rubioideae (Andersson 1996; Bremer 1996; Bremer & Thulin 1998; Andersson & Rova 1999). The two closely related tribes Urophylleae and Pauridiantheae are strong Al accu- mulators. Robbrecht (1988) placed both tribes in a position between Isertieae (Cin- chonoideae) and Hedyotideae (Rubioideae), maintaining them in the Cinchonoideae

Downloaded from Brill.com09/24/2021 04:57:17PM via free access Jansen, Robbrecht, Beeckman & Smets — Al accumulation in Rubiaceae 205 on account of the exotestal cell anatomy matching the Isertieae. This placement is reinforced by data obtained by Manen and Natali (1996) who reported that the cpDNA deletion characteristic of Rubioideae is not present in Urophyllum. Bremer (1996: 45) has associated Pauridiantha with the Al accumulator Lasianthus. She pointed out that there are morphological similarities between the two genera (habit, shape, ), but there are major differences between the gynoecia of Lasianthus (4–12 locular ovaries, each with one ) and the Pauridiantheae (2–4- carpellate with false septa and U-shaped placentas with many ; Bangoura 1993). The recent cladograms of Bremer and Thulin (1998) and Andersson and Rova (1999) show a basal position of Pauridiantha and Urophyllum within the Rubioideae subfamily. The presence of Al accumulation corroborates this position. There are a few other representatives of the Cinchonoideae which show aluminium accumulation in their leaves. Since for most genera only one specimen is found to be positive, these records are probably due to misidentification of the material used by Chenery (1948b): Cinchona (1/4) and Remijia (1/5) (Cinchoneae), Mussaendopsis (1/2) (Coptosapelteae), and Acrobotrys (1/1) (Rondeletieae). The results of Copto- sapelta (7/7), however, are remarkable and probably correct, although we were un- able to demonstrate Al accumulation in the wood of this genus. Robbrecht (1994: 175) already remarked that the position of Coptosapelta is problematic. The combi- nation of Al accumulation in leaves and raphides (Metcalfe & Chalk 1950; Fukuoka 1980) points to a position in the Rubioideae, but this is contradicted by the contorted corolla aestivation and the occurrence of secundary pollen presentation. Al accumulation is not observed in wood of Ixoroideae. Besides a majority of ge- nera which were always negative, Chenery (1948b) obtained positive leaf tests for a few species of the genera Lamprothamnus (1/1), Pavetta (3/37), and Scyphostachys (1/1). It is clear that in this subfamily Al accumulators are (almost) completely ab- sent. The Knoxieae have been associated with other tribes with solitary ovules in the Antirheoideae, as well as traditionally (Schumann 1891: 16: supertribe Guettardinae) as in Robbrechtʼs (1994) classification system of the Rubiaceae. Evidence presented here supports a placement in the Rubioideae, a position advocated by Verdcourt (1958) and Bremekamp (1966), since leaves and wood of Calanda, Knoxia and Pentanisia frequently accumulate Al. Only two wood samples of Chlorochorion reacted nega- tively, but it is possible that Al accumulates in the leaves of this genus. Note that an affinity with the Rubioideae–Hedyotideae has also been supported by Bremer (1996), who proposed to merge Hedyotideae, Knoxieae and Spermacoceae into one tribe Hedyotideae. Al accumulation in the monogeneric Craterispermeae allows similar considera- tions. Despite heterostylous and the presence of raphides, Craterispermum was concluded to occupy a rather isolated position in the Antirheoideae (Robbrecht 1988; Igersheim 1992). Taking the strong Al accumulation into account, Verdcourtʼs (1958) placement near the Psychotrieae seems justified. Wood anatomy also corrobo- rates this. Axial parenchyma bands, a feature rare in Rubiaceae, have been observed in the wood of Craterispermum and are also present in Colletoecema, Morinda,

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Gaertnera, and (Jansen et al. 1996, 1997). Another similarity with these genera is the presence of fibre tracheids (non-septate fibres with bordered pits on radial and tangential walls). Al accumulation in leaves is also reported in a single specimen of Pachystigma (1/4) and Perakanthus (1/2) (Vanguerieae), and Asemnantha (1/1) (Chiococceae). The positive test for leaves of Alberta (2/2) (Alberteae) and Canthium (14/34) (Vangue- rieae) concern a larger number of species and need verification. While Chenery (1948b) reports 14 positive tests for Canthium (14/25), all specimens of Canthium tested by Webb (1954) are negative (9/0). Canthium is a highly diverse assemblage (Bridson 1992) and it would be interesting to know if the species tested for leaf content by Chenery (1948b) really belong to what would now be considered as Canthium. Al accumulation in Rubiaceae is almost limited to the Rubioideae; all 29 genera that gave a positive chrome azurol-S test are associated with this subfamily according to recent intrafamilial classification. Moreover, 793 specimens (95%) of all 830 Rubiaceae that accumulate Al in their leaves belong to the Rubioideae (including Craterispermeae, Knoxieae, Pauridiantheae, and Urophylleae). The Psychotrieae and associated Coussareeae, and Prismatomerideae are the core group of tribes where accumulation occurs, and probably also where it is most strongly expressed. These tribes are essentially the woody, uniovulate and fleshy-fruited representatives of the Rubioideae. Among the woody Rubioideae, the absence of Al accumulation in wood of the Morindeae s.str. is striking. It supports the recent segregation of the Prismato- merideae (Igersheim & Robbrecht 1994) from the Morindeae. However, leaves of some Morindeae do accumulate Al. The more herbaceous, dry-fruited groups of the Rubioideae are probably more derived, although most but not all tribes have multiovulate . It seems that they have no pronounced Al accumulation: Schradereae, Argostemmateae, Hedy- otideae p.p., and Ophiorhizeae. Uniovulate predominantly herbaceous tribes in the Rubioideae with very few aluminium accumulators are Rubieae, Spermacoceae, Antho- spermeae, and Paederieae. The herbaceous genus Coccocypselum (Coccocypseleae) is a strong accumulator. It is interesting to note that Bremer (1996) found a highly supported relationship between this genus and the woody genus Faramea, which is also a strong accumula- tor. Another remarkable accumulator is the genus Danais (Cinchoneae/Hedyotideae) which includes multiovulate woody climbers; 17 of the 18 species studied by Chenery (1948b) proved positive. The wood samples tested here confirm that this genus strong- ly accumulates Al. Danaisʼ original position was in the Cinchoneae (Schumann 1891), but Bremekamp (1952) and Bremer (1996) included it in the Rubioideae. A minor comment should be made on the Al accumulation in Saprosma (Paederieae). The genus has traditionally been placed in the Psychotrieae, but Puff (1992) proposed its transfer to the Paederieae. The fact that all specimens of Saprosma tested by Chenery (1946, 1948a, b) are positive puts some doubt on this transfer, since Al accumulation is not found in other genera of the Paederieae. Our chrome azurol-S tests of Saprosma were positive, although only the outermost part of the wood in contact with the bark was positive.

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The evolution of Al accumulation in Rubiaceae A plot of our data on a cladogram which is based on rbcL sequences (Bremer & Thulin 1998) shows the concentration of Al accumulation in Rubioideae and its oc- currence in the basalmost of this group (see Figure 1 on the next page; with regard to the uncertain records in Sabicea and Cinchona, see above). The taxonomic restriction of the feature to certain taxa demonstrates that the character is probably due to evolutionary (genetic) control of metabolic pathways. Hence, we suggest that Al accumulation has evolved in an ancestor of the Rubioideae, and not in other sub- families. Because representatives of all subfamilies of Rubiaceae frequently grow next to each other in tropical rain forests, it is possible that Cinchonoideae and Ixoroideae have other mechanisms at their disposal to tolerate Al stress. Other mecha- nisms are for instance Al-chelation (Foy et al. 1978; Cuenca et al. 1990, 1991; Lüttge 1997), exclusion by impermeability of the endodermal cells to Al3+ (Cuenca & Herrera 1987; Cuenca et al. 1990), or alkalinisation of the rhizosphere, which diminishes Al-mobility and avoids the stress (Lüttge 1997). The phylogeny of the Rubioideae was recently also estimated from sequence vari- ation in the rps16 intron (cpDNA) by Andersson and Rova (1999). The three basal- most clades in their cladogram consist of the Pauridiantheae, Urophylleae, Ophi- orrhizeae, Raritebe (Isertieae), Lasianthus (?Psychotrieae), (Perameae), Coussareeae, Coccocypselum (Coccocypseleae), Cruckshanksieae, Declieuxia (Psy- chotrieae), and (Cinchoneae/Hedyotideae). Except for the genera Ophiorrhiza and Hindsia, most of these taxa are strong Al accumulators. It would be interesting to verify Al accumulation in leaves of the herbaceous tribe Cruckshancksieae, and the genus Raritebe, since these basal taxa have not been tested before. In the two other clades of the Rubioideae, the Al accumulating tribe Knoxieae is rather basal, and the more derived clades show a mixed occurrence of the character. Chenery and Sporne (1976) concluded that accumulation is a primitive character- istic because Al accumulation is statistically correlated with seven primitive charac- ters listed by Sporne (1969). If it is true that the woody tribes of the Rubioideae are considered to be more primitive, the postulated primitiveness of Al accumulation is at first sight corroborated by the situation in this subfamily, since the woody tribes gen- erally are accumulators. Not only in Al accumulation, but also in other characters which are probably re- lated to specific metabolic pathways, the subfamily Rubioideae clearly differs from other rubiaceous groups. Examples are the presence of raphides (Robbrecht 1988), anthraquinones (Young et al. 1996), and due to a deletion a loss of one of the two atpB promotors in the cpDNA (Manen & Natali 1996). Therefore, the segregation of the Rubioideae from the rest of the Rubiaceae might be one of the earliest events in the evolutionary history of the family.

Influence of environmental factors on Al accumulation Al is the classic example of an element whose uptake is being influenced by pH conditions. Al accumulators have a worldwide distribution, but they occur particu-

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Ligustrum (Oleaceae) other RU Amphidasya ISE (L 1/1) Pauridiantha PAU (W 4/6; L 5/5) ARG (L 0/x) Ophiorrhiza OPH (W 0/1; L 0/x) Lasianthus PSY (W 7/9; L 61/62) Coccocypselum COC (L 27/27) Coussarea COU (W 11/11; L 8/8) Faramea COU (W 13/13; L 83/83) Morinda MOR (W 0/12; L 8/22) Schradera SCH (W 0/3; L 3/7) Hydnophytum PSY (L 3/18) Psychotria PSY (W 11/36; L 207/366) ARG (L 0/x) Mycetia ISE (W 0/1; L 1/1) Oldenlandia HED (W 0/1; L 5/13) HED (W 0/6; L 0/x) Rubia RUB (W 0/1; L 1/4) THE (L 0/x) ANT (W 0/3; L 0/1) AL CAL (W 0/1; L 0/x) Pogonopus CON (L 0/x) HIP (L 0/1) HIP (L 0/x) Heinsia ISE (W 0/2; L 0/x) ISE Mussaenda ISE (W 0/2; L 0/2) Pseudosabicea ISE Sabicea ISE (W 0/2; L 1/1) Tamridaea (new genus; Bremer & Thulin 1998) HED - now Sabiceeae (W 0/1; L 0/1) VAN (W 0/1) VAN (L 0/x) PAV (W 0/3; L 0/9) GAR (W 0/5; L 0/9) GAR (W 0/2; L 0/3) COF (W 0/14; L 0/x) ISE (W 0/1) GAR (L 0/x) GUE (W 0/1; L 0/3) ISE (W 0/1; L 0/x) GUE (L 0/x) GUE (W 0/1; L 0/2) HED (L 0/x) RON (W 0/1; L 0/x) HIL (W 0/1; L 0/x) Hamelia HAM (W 0/6; L 0/x) HAM (L 0/x) Cinchona CIN (W 0/1; L 1/4) CIN (W 0/2; L 0/x) Isertia ISE (W 0/1; L 0/x) Cephalanthus CEP (W 0/2; L 0/x) NAU (L 0/2) COP (W 0/2; L 0/3) POR (W 0/1; L 0/x) CHI (W 0/2; L 0/x) CHI (W 0/2; L 0/x) CAT (L 0/x) POR

Downloaded from Brill.com09/24/2021 04:57:17PM via free access Jansen, Robbrecht, Beeckman & Smets — Al accumulation in Rubiaceae 209 larly in tropical rain forests, preferably on acid soils (pH 3 –5), and they are almost absent from dry areas (Larcher 1980). Accordingly, accumulation is a process that depends partly on the influence of heredity and partly on ecological conditions. We believe that accumulators in Rubiaceae are from tropical regions, and we also suggest that the lack of the feature in the more derived and herbaceous Rubioideae (Anthospermeae, Hedyotideae p.p., Paederieae, Rubieae, Spermacoceae) is possibly related to their adaptation to more xeric, alkaline soils and their distribution into more temperate regions. Unfortunately, precise environmental conditions are not available and are beyond the scope of the present study.

Fruit and flower colours in Rubiaceae and their relation to Al accumulation Chenery (1946, 1948a) stated that a very high correlation exists between the pres- ence of Al in abnormal quantities and a bright blue colour of the of dicotyle- dons. Blue fruits are rather exceptional in Rubiaceae, apart from taxa which belong to the Rubioideae: Cephaelis, Trichostachys, Lasianthus, and Coccocypselum. Some genera (e.g. Psychotria and Geophila) have both red- and blue-fruited species. As these genera are Al accumulators, the correlation between Al accumulation and blue-fruited plants seems to be rather positive for the Rubiaceae. During Cheneryʼs search for Al accumulators, some plants were also found to have flowers varying from pink to blue like the flower colour in the popular French Hortensia macrophylla DC. (Allen 1943). White corollas are dominant in Rubia- ceae, but blue or bluish corollas have evolved in many herbaceous taxa, e.g. in all members of the tribe Knoxieae, in many Hedyotideae and in a few large tropical woody genera such as Faramea and Palicourea (Robbrecht 1988). Genera with bluish corollas have at least some species that are Al accumulators (e.g. Pentanisia, Sacosperma, Manettia, Spermacoce, Palicourea and Faramea). Gottsberger and Gottlieb (1981) mentioned that the driving force to complex formation of blue col- our pigments in angiosperms is partly attributed to the presence of Al3+, but also to other metal ions as K+, Mg2+, Fe3+, and Mo3+. Thus, not all blue-fruited plants are Al accumulators. Comparison of the list of families that accumulate Al with the fam- ilies having blue flowers (Gottsberger & Gottlieb 1980, 1981) shows that there is no correlation at a high taxonomic level; many blue-flowered monocots and families such as Bromeliaceae, Dipsacaceae, Campanulaceae, Boraginaceae, and Plumbagina- ceae do not accumulate Al.

Fig. 1. Strict consensus of Bremer and Thulin (1998) based on rbcL sequences. Genera in bold have at least one Al accumulator; genera in regular font style do not accumulate Al; genera in italic were not tested. Tribal positions are indicated by a three-letter suffix corre- sponding to the tribes in Robbrecht (1994). The nominator indicates the number of Al accu- mulating specimens, the denominator gives the total number of specimens tested; W = chrome azurol-S test; L = Al test for leaves; x = number of specimens tested not known; RU = sub- family Rubioideae; AL = other subfamilies of Rubiaceae.

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CONCLUSION

A positive chrome azurol-S test is found in 103 specimens (28 genera) which are all included in the Rubioideae according to recent subfamilial insights of the Rubiaceae. Our results agree well with earlier wood data and with previous records of Al accu- mulation in rubiaceous leaves. Al is shown to accumulate more strongly in leaves than in secondary xylem. The major conclusion is that Al accumulation in Rubiaceae can be used as an ancillary criterion to evaluate the systematic relationships of groups of dubious affinity. In particular, the ability to accumulate Al supports the return of the tribes Craterispermeae, Knoxieae, Pauridiantheae and Urophylleae to the Rubi- oideae, because the character is more or less restricted to this subfamily. The lack of the character in the more herbaceous and derived taxa is possibly associated with their tendency to herbaceousness and/or their distribution into more xeric, alkaline soils and more temperate conditions. Finally, more rigorous analytical work will pro- vide more precise data on the quantification of the Al level and these are needed for objective categorisation of the groupings, statistical analyses, or ecological purposes.

ACKNOWLEDGEMENTS

Thanks are due to Anja Vandeperre and Sheng-Sheng Huang for assisting the chrome azurol-S tests. We are grateful to Prof. Dr. P. Baas (Leiden University, The Netherlands), Mr. P. Détienne (CIRAD- Forêt, Montpellier, France), Dr. J. Koek-Noorman (Utrecht University, The Netherlands), and Dr. R.B. Miller (U.S. Forest Products Laboratory, Madison, U.S.A.) for the supply of wood samples. The curators of the herbaria of Kew (K), Leiden (L), Missouri (MO), Paris (P), and Wageningen (WAG) are acknowledged for the permission to use small wood samples of herbarium material. Steven Jansen holds a scholarship of the Research Council of the K.U. Leuven. This research is sup- ported by a grant from the Research Council of the K.U. Leuven (OT/97/23) and by grants from the Fund for Scientific Research – Flanders (F.W.O., Belgium): project number G. 0143.95.

REFERENCES

Allen, R.C. 1943. Influence of aluminum on the flower color of DC. Contr. Boyce Thompson Inst. 13: 221–242. Andersson, L. 1996. Circumscription of the tribe Isertieae (Rubiaceae). In: E. Robbrecht, C. Puff & E. Smets (eds.), Second International Rubiaceae Conference, Proceedings. Opera Bot. Belg. 7: 139–164. Andersson, L. & J.H.E. Rova. 1999. The rps16 intron and the phylogeny of the Rubioideae (Rubiaceae). Plant Syst. Evol. 214: 161–186. Bangoura, D. 1993. Ovary structure and in the tribe Pauridiantheae (Rubiaceae). Bull. Jard. Bot. Nat. Belg. 62: 419–428. Bremekamp, C.E.B. 1952. The African species of Oldenlandia L. sensu Hiern et K. Schumann. Verh. Kon. Ned. Akad. Wet., Serie 2, 18: 1–297. Bremekamp, C.E.B. 1966. Remarks on the position, the delimitation and the subdivision of the Rubiaceae. Acta Bot. Neerl. 15: 1–33. Bremer, B. 1996. Phylogenetic studies within Rubiaceae and relationships to other families based on molecular data. In: E. Robbrecht, C. Puff & E. Smets (eds.), Second International Rubiaceae Conference, Proceedings. Opera Bot. Belg. 7: 33–50.

Downloaded from Brill.com09/24/2021 04:57:17PM via free access Jansen, Robbrecht, Beeckman & Smets — Al accumulation in Rubiaceae 211

Bremer, B. & M. Thulin. 1998. Collapse of Isertieae, re-establishment of Mussaendeae, and a new genus of Sabiceeae (Rubiaceae); phylogenetic relationships based on rbcL data. Plant Syst. Evol. 211: 71–92. Bridgwater, S. & P. Baas. 1982. Wood anatomy of Xanthophyllum Roxb. IAWA Bull. n.s. 3: 115–125. Bridson, D.M. 1992. The genus Canthium (Rubiaceae–Vanguerieae) in tropical Africa. Kew Bull. 47: 353–401. Chenery, E.M. 1946. Are Hydrangea flowers unique? Nature 158: 240–241. Chenery, E.M. 1948a. Aluminium in plants and its relation to plant pigments. Ann. Bot. 12: 121–136. Chenery, E.M. 1948b. Aluminium in the plant world. Part I, General survey in dicotyledons. Kew Bull. 1948: 173–183. Chenery, E.M. 1949. Aluminium in the plant world. Part II, Monocotyledons and gymn- osperms. Part III, Cryptograms. Kew Bull. 4: 463–473. Chenery, E.M. & K.R. Sporne. 1976. A note on the evolutionary status of aluminium-accumu- lators among dicotyledons. New Phytol. 76: 551–554. Cuenca, G. & R. Herrera. 1987. Ecophysiology of aluminium in terrestrial plants, growing in acid and aluminium-rich tropical soils. Ann. Soc. R. Zool. Belg. 117, Suppl. 1: 57–74. Cuenca, G., R. Herrera & E. Medina. 1990. Aluminium tolerance in of a tropical cloud forest. Plant Soil 125: 169–175. Cuenca, G., R. Herrera & T. Merida. 1991. Distribution of aluminium in accumulator plants by X-ray microanalysis in Richeria grandis Vahl leaves from a cloud forest in . Plant Cell Environ. 14: 437–441. Ernst, W. 1972. Ecophysiological studies on heavy metal plants in south Central Africa. Kirkia 8: 125–145. Foy, C.D., R.L. Chaney & M.C. White. 1978. The physiology of metal toxicity in plants. Annu. Rev. Plant Physiol. 29: 511–566. Fukuoka, N. 1980. Studies in the floral anatomy and morphology of Rubiaceae IV. Rondeletieae and Cinchoneae. Acta Phytotaxon. Geobot. 31: 65–71. Gottsberger, G. & O.R. Gottlieb. 1980. Blue flowers and phylogeny. Rev. Bras. Bot. 3: 79–83. Gottsberger, G. & O.R. Gottlieb. 1981. Blue flower pigmentation and evolutionary advance- ment. Biochem. Syst. Ecol. 9: 13–180. Hutchinson, G.E. 1945. Aluminum in soils, plants, and animals. Soil Sci. 60: 29–40. Hutchinson, G.E. & A. Wollack. 1943. Biological accumulators of aluminum. Conn. Acad. Arts and Sci. Trans. 35: 73–128. IAWA Committee. 1989. IAWA list of microscopic features for hardwood identification. IAWA Bull. n.s. 10: 219–332. Igersheim, A. 1992. The ovary, and seed development of Craterispermum (Rubiaceae). Belg. J. Bot. 125: 101–113. Igersheim, A. & E. Robbrecht. 1994. The character states and relationships of the Prismato- merideae (Rubiaceae–Rubioideae). Comparison with Morinda and comments on the cir- cumscription of the Morindeae s.str. In: E. Robbrecht (ed.), Advances in Rubiaceae macrosystematics. Opera Bot. Belg. 6: 61–79. Jansen, S., E. Robbrecht, H. Beeckman & E. Smets. 1996. Gaertnera and Pagamea: genera within the Psychotrieae or constituting the tribe Gaertnereae? A wood anatomical and palynological approach. Bot. Acta 109: 466–476. Jansen, S., E. Robbrecht, H. Beeckman & E. Smets. 1997. Wood anatomy of the predomi- nantly African representatives of the tribe Psychotrieae (Rubiaceae–Rubioideae). IAWA J. 18: 169–196.

Downloaded from Brill.com09/24/2021 04:57:17PM via free access 212 IAWA Journal, Vol. 21 (2), 2000

Keating, R.C. & V. Randrianasolo. 1988. The contribution of leaf architecture and wood anat- omy to classification of the Rhizophoraceae and Anisophylleaceae. Annals Missouri Bot. Gard. 75: 1343–1368. Kukachka, B.F. & R.B. Miller. 1980. A chemical spot-test for aluminium and its value in wood identification. IAWA Bull. n.s. 1: 104–109. Larcher, W. 1980. Physiological plant ecology. Ed. 2. Springer-Verlag, Berlin, etc. Lüttge, U. 1997. Physiological ecology of tropical plants. Springer-Verlag Berlin, etc. Manen, J.-F. & A. Natali. 1996. The chloroplast atpB-rbcL spacer in Rubiaceae. In: E. Robbrecht, C. Puff & E. Smets (eds.), Second International Rubiaceae Conference, Proceedings. Opera Bot. Belg. 7: 51–57. Metcalfe, C.R. & L. Chalk. 1950. Anatomy of the dicotyledons. Clarendon Press, Oxford. Metcalfe, C.R. & L. Chalk. 1983. Anatomy of the dicotyledons. Vol. II. Wood structure and conclusion of the general introduction. Ed. 2. Clarendon Press, Oxford. Moomaw, J.C., M.T. Nakamura & G.D. Sherman. 1959. Aluminium in some Hawaiian plants. Pac. Sci. 8: 335–341. Puff, C. 1992. On the correct tribal position of Saprosma Bl. (Rubiaceae). In: Second Flora Malesiana Symposium. Programme and summary of papers and posters: 34. Quirk, J.T. 1980. Wood anatomy of the Vochysiaceae. IAWA Bull. n.s. 1: 172–179. Robbrecht, E. 1988. Tropical woody Rubiaceae. Opera Bot. Belg. 1: 1–271. Robbrecht, E. 1994. Supplement to the 1988 outline of the classification of the Rubiaceae. Index to genera. In: E. Robbrecht (ed.), Advances in Rubiaceae macrosystematics. Opera Bot. Belg. 6: 173–196. Robbrecht, E., C. Puff & E. Smets (eds.). 1996. Second International Rubiaceae Conference, Proceedings. Opera Bot. Belg. 7. Robinson, W.O. & G. Edgington. 1945. Minor elements in plants, and some accumulator plants. Soil Sci. 60: 15–28. Rogers, G.K. 1981. The wood anatomy of Gleasonia, Henriquezia, and Platycarpum (Rubiaceae) and its bearing on their classification: some new considerations. Brittonia 33: 461–465. Rogers, G.K. 1984. Gleasonia, Henriquezia, and Platycarpum (Rubiaceae). Flora Neotropica monograph 39. New York. Schumann, K. 1891. Rubiaceae. In: A. Engler & K. Prantl (eds.), Die natürlichen Pflanzen- familien 4. Engelmann, Leipzig. Sporne, K.R. 1969. The ovule as an indicator of evolutionary status in angiosperms. New Phytol. 68: 555. Verdcourt, B. 1958. Remarks on the classification of the Rubiaceae. Bull. Rijksplantentuin Bruss. 28: 209–281. Webb, L.J. 1954. Aluminium accumulation in the Australian– flora. Aust. J. Bot. 2: 176–197. Young, M.C.M, M.R. Braga, S.M.C. Dietrich, V.S. Bolzani, L.M.V. Trevisan & O.R. Gottlieb. 1996. Chemosystematic markers of Rubiaceae. In: E. Robbrecht, C. Puff & E. Smets (eds.), Second International Rubiaceae Conference, Proceedings. Opera Bot. Belg. 7: 205–212.

Downloaded from Brill.com09/24/2021 04:57:17PM via free access