IAWA Journal, Vol.

21 (2), 2000: 197–212

ALUMINIUM ACCUMULATION IN RUBIACEAE:

AN ADDITIONAL CHARACTER FOR THE DELIMITATION OF THE
SUBFAMILY RUBIOIDEAE?
by

S. Jansen 1, E. Robbrecht 2, H. Beeckman3 & E. Smets 1

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 leaves 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, Coussarea, Faramea, and Rudgea. 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 Anthospermeae, Paederieae,
Rubieae, and Spermacoceae. Al accumulation in the Urophylleae, Pauri-
diantheae, Craterispermeae, and Knoxieae supports earlier associations
of these tribes with the Rubioideae.
Key words: Aluminium accumulation, chrome azurol-S test, chemo-
taxonomy, 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 Plant Systematics, Institute of Botany 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 Africa, Leuvensesteenweg 13, B-3080 Tervuren, Belgium.
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 plants inhabiting tropical or subtropical regions. Above the family
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 species 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 Ophiorrhizeae
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 Rubia-
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
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 herbarium 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 Cinchonoideae sensu Robbrecht, positive specimens occurred in the
tribes Pauridiantheae (Pauridiantha, Poecilocalyx, and Stelecantha) and Urophylleae
(Leucolophus, Maschalocorymbus, Praravinia, and Urophyllum). No accumulators
were found in the subfamily Ixoroideae. In the Antirheoideae sensu Robbrecht, posi-
tive taxa were restricted to the monogeneric tribe Craterispermeae, and some genera
of the Knoxieae, namely Calanda, Knoxia and Pentanisia. As for the subfamily Rubi-
oideae, many wood samples reacted positively. We observed high frequencies for
accumulation in the neotropical genera Psychotria 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. Danais (3/4), Hedyotis
(3 /3), Lasianthus (7 /9), Prismatomeris (4 /5), Rennellia (3 /3), Saprosma (2 /2), Tri-
chostachys (2 /2), and the single specimen tested of Margaritopsis (1 /1). In contrast,
only few representatives of Colletoecema (2 /6), Gaertnera (1/ 4), Morinda (1/6),
Palicourea (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 (Craterispermum cerinanthum, Faramea occidentalis, Lasianthus batangen-
sis, Psychotria vasiviensis). In contrast, the wood samples of Colletoecema dewevrei,
Maschalocorymbus corymbosus, Otomeria micrantha, Pentanisia schweinfurthii, and
Psychotria peduncularis differed in their reaction; the tests gave negative as well as
positive or intermediate results for these species.
200 IAWA Journal, Vol. 21 (2), 2000

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
Cinchoneae Cinchona (0/1); Ferdinandusa5 Cinchona (1/4)3;
Ferdinandusa (0/5); Ferdinandusa3;
Remijia (0/1); Remijia (1/5)3;
3 other genera negative 6 other genera negative
Calycophylleae 2 genera negative 4 genera negative
Coptosapelteae Coptosapelta (0/1); Coptosapelta (7/7)3;
Mussaendopsis (0/1); Mussaendopsis (1/2)3;
5 other genera negative 6 other genera negative
Naucleeae Gyrostipula 5 genera negative
Hillieae 3 genera negative 2 genera negative
Henriquezieae Henriquezia (0/1); Henriquezia5, 6; Henriquezia3; Platycarpum3
Gleasonia (0/1); Gleasonia6 (3/3);
Platycarpum (0/1) Platycarpum6 (1/4)
Rondeletieae 7 genera negative Acrobotrys (1/1)3;
9 other genera negative
Simireae Simira Simira3
Sipaneeae Sipanea Sipanea3
Condamineeae Chimarrhis 5 genera negative
Portlandia group Coutarea, Exostemma 9 genera negative
Isertieae Mycetia (0/1); Amphidasya (1/1)3;
Sabicea (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)
→
Jansen, Robbrecht, Beeckman & Smets — Al accumulation in Rubiaceae 201

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

IXOROIDEAE
Gardenieae 30 genera negative 29 genera negative
Pavetteae Pavetta (0/3); Pavetta (3/37)3;
13 other genera negative 6 other genera negative
Coffeeae 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 Retiniphyllum
Vanguerieae Canthium (0/1); Canthium (14/34)3, 4;
Pachystigma (0/1); Pachystigma (1/4)3;
4 other genera negative Perakanthus (1/2)3;
6 other genera negative
Guettardeae 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 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 Manettia (2/5)2,3;
4 other genera negative
Hedyotideae Carphalea (1/2); Gouldia5 (probably Hedyotis (11/14)3, 4;
Hedyotis (3/3); Hedyotis) Oldenlandia (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 Synaptantha (1/3)3, 4;
17 other genera negative
Ophiorrhizeae Ophiorrhiza Ophiorrhiza3, Spiradiclis3
Coccocypseleae (herbaceous) Coccocypselum (27/27)1, 2, 3
Argostemmateae (herbaceous) Argostemma3, Neurocalyx3
Hamelieae Hamelia 4 genera negative
→
202 IAWA Journal, Vol. 21 (2), 2000

Subfamily / Tribe I: Own chrome II: Chrome azurol-S III: Al tests on leaves
azurol-S tests tests from literature
(Rubioideae ctd)
Schradereae Schradera (0/3) Schradera (3/7)3;
Lecananthus3, Leucocodon3
Psychotrieae Chassalia (0/2); Psychotria5 Amaracarpus (8/11)2, 3;
Declieuxia (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 Geophila (2/8)2, 3, 4;
Hedstromia (1/1)3;
Hydnophytum (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;
Saldinia (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 Gynochthodes (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 Mitchella Damnacanthus (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 Coprosma (0/5); Coprosma (1/11)2, 3, 4;
6 other genera negative 10 other genera negative
Spermacoceae Diodia (0/1); Emmeorhiza (1/1)3;
Emmeorhiza (0/1); Richardia (2/9)3, 4;
Spermacoce (0/4); Spermacoce (10/18)1, 2, 3, 4;
Staelia (0/1) Staelia (1/1)3;
6 other genera negative
Rubieae Crucianella (0/1); Galium (2/12)3; Rubia (1/4)3;
Rubia (0/1) 8 other genera negative
inc. sed. 3 genera negative 16 genera negative
Jansen, Robbrecht, Beeckman & Smets — Al accumulation in Rubiaceae 203

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 Gillespiea, 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 of Ferdinandusa 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, leaf 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.
204 IAWA Journal, Vol. 21 (2), 2000

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 genus 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 Isertia (Isertieae) and
Heinsia and Mussaenda (Mussaendeae), 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
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, flower shape,
inflorescences), but there are major differences between the gynoecia of Lasianthus
(4–12 locular ovaries, each locule with one basal ovule) and the Pauridiantheae (2– 4-
carpellate with false septa and U-shaped placentas with many ovules; 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 flowers 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,
206 IAWA Journal, Vol. 21 (2), 2000

Gaertnera, and Pagamea (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 ovary locules. 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.
Jansen, Robbrecht, Beeckman & Smets — Al accumulation in Rubiaceae 207

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 clades 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 Al 3+ (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), Perama (Perameae),
Coussareeae, Coccocypselum (Coccocypseleae), Cruckshanksieae, Declieuxia (Psy-
chotrieae), and Hindsia (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-
208 IAWA Journal, Vol. 21 (2), 2000

Ligustrum (Oleaceae)
other Gentianales
RU Amphidasya ISE (L 1/1)
Pauridiantha PAU (W 4/6; L 5/5)
Neurocalyx 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)
Argostemma ARG (L 0/x)
Mycetia ISE (W 0/1; L 1/1)
Oldenlandia HED (W 0/1; L 5/13)
Pentas HED (W 0/6; L 0/x)
Rubia RUB (W 0/1; L 1/4)
Theligonum THE
Nertera ANT (L 0/x)
Anthospermum ANT (W 0/3; L 0/1)
AL Calycophyllum CAL (W 0/1; L 0/x)
Pogonopus CON (L 0/x)
Hippotis HIP (L 0/1)
Pentagonia HIP (L 0/x)
Heinsia ISE (W 0/2; L 0/x)
Pseudomussaenda 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)
Virectaria HED - now Sabiceeae (W 0/1; L 0/1)
Meyna VAN (W 0/1)
Vangueria VAN (L 0/x)
Ixora PAV (W 0/3; L 0/9)
Gardenia GAR (W 0/5; L 0/9)
Bertiera GAR (W 0/2; L 0/3)
Coffea COF (W 0/14; L 0/x)
Aoranthe ISE (W 0/1)
Mitriostigma GAR (L 0/x)
Antirhea GUE (W 0/1; L 0/3)
Gonzalagunia ISE (W 0/1; L 0/x)
Chomelia GUE (L 0/x)
Guettarda GUE (W 0/1; L 0/2)
Rachicallis HED (L 0/x)
Rondeletia RON (W 0/1; L 0/x)
Hillia HIL (W 0/1; L 0/x)
Hamelia HAM (W 0/6; L 0/x)
Hoffmannia HAM (L 0/x)
Cinchona CIN (W 0/1; L 1/4)
Ladenbergia CIN (W 0/2; L 0/x)
Isertia ISE (W 0/1; L 0/x)
Cephalanthus CEP (W 0/2; L 0/x)
Nauclea NAU (L 0/2)
Uncaria COP (W 0/2; L 0/3)
Exostema POR (W 0/1; L 0/x)
Erithalis CHI (W 0/2; L 0/x)
Chiococca CHI (W 0/2; L 0/x)
Catesbaea CAT (L 0/x)
Cubanola POR
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 fruits 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
Hydrangea 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+, Mg 2+, Fe 3+, 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 tree 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.
210 IAWA Journal, Vol. 21 (2), 2000

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.

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