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Biochemical systematics and ecology of the toddalioideae in the central part of the West African forest zone
BiochemicalSystematicsandEcology.Vol.6, pp. 239to 245. © PergamonPressLtd.1978.Printedin England.
0305-1978/78/0901--0239502.00/0
Biochemical Systematics and Ecology of the Toddalioideae in the Central Part of the West African Forest Zone* PETER G. WATERMAN and IBRAHIM A. MESHAL and J. B. HALL and MICHAEL D. SWAINE Department of Pharmaceutical Chemistry, University of Strathclyde, Glasgow G1 1XW, Scotland Department of Botany, University of Ghana, Legon, Ghana Key Word I n d e x - - Araliopsis soyauxii: Diphasia angolensis; Oricia suaveolens; Teclea verdoomiana; Vepris heterophylla; Rutaceae;Toddalioideae; alkaloids; triterpenes; chemotaxonomy; chemical ecology. Abstract--The distribution, ecology and secondary metabolism of five species of Rutaceae (subfamily Toddalio-
ideae), Araliopsis soyauxii, Diphasia angolensis, Oricia suaveolens, Teclea verdoomiana, and Vepris heterophylla, occurring within the central part of the West African ForestZone is reviewed. It is demonstratedthat the five species exhibit niche separationin such a way that no two specieswith similar complementsof secondarymetabolitesappear to be in direct competition. It is suggested,in support of Janzen's hypothesis,that this situation may haveevolved to alleviate pest pressure. The chemical variation between Ghanaian and Nigerian populations of O. suaveolens is discussed in the light of observations on their ecology. Possiblephylogenetic relationships between these species are discussed. Introduotion
Results and discussion
The Rutaceae is divided into three major subfamilies, Rutoideae, Toddalioideae and Aurantioideae plus four minor, often disputed taxa [1]. Engler [2] recognised eight genera of Toddalioideae in Tropical Africa. These comprise shrubs and trees characterised by digitate leaves with 3-5 leaflets, small, generally unisexual flowers in paniculate inflorescences, and drupaceous fruits. The genera are distinguished, rather artificially, by the number of stamens (equal to or twice the number of petals), number of carpels (1, 2, or 4-5), number of ovules per carpel (1 or 2), and degree of fusion of fruiting carpels. Keay [3] in his revision of the West African species stated that the limits between these genera needed to be thoroughly revised, and this opinion was endorsed by Voorhoeve [4]. The problem is exemplified by Letouzey [5] who includes in his revision of Rutaceae from Gabon a number of taxa of Toddalioideae which, because of incomplete knowledge of flowers of one sex, or of fruits, cannot be ascribed with certainty to any genus.
Five genera have been recorded in the area considered in the present paper (Ivory Coast eastwards to the 5 ° E. meridian--Fig. 1), represented by five species: Araliopsis soyauxii Engl. (which we consider to include A. tabouensis Aubr6v. ~t Pellegr.) extending from Liberia to Gabon; Diphasia angolensis (Hiern) Verdoorn (synonym D. k/aineana Pierre [6]) from Guinde to Angola; Oricia suaveolens (Engl.) Verdoorn from Guinde to Zafre; Teclea verdoorniana Exell Et Mendonqa from Sierre Leone to Zafre; and Vepris heterophylla (Engl.) Letouzey (previously known as Teclea sudanica A. Chev. [7]) from Ghana to Mali and Cameroun. They can be distinguished by the key given in Table 1. According to Engler's classification [2], Teclea is placed in the subtribe Amyridinae, distinguished by a unilocular ovary; Diphasla and Oricia in the Oriciinae, distinguished by 2 - 4 incompletely connate carpels, stamens usually twice the number of petals, and endosperm usually present in the seed; and Araliopsis and Vepris (together with the other ° Part X in the series "'Chemosystematics in the African genera, Toddalia, Toddaliopsis and Rutaceae". For Part IX see Waterman, P. G., and Gray, Oriciopsis) into the Toddaliinae characterised A. I. (1978) Phytochemistry 17, 845. by 2 - 5 completely connate carpels, stamens (Received for publication 10 January 1978) 239
240
PETER G. WATERMAN, IBRAHIM A. MESHAL. J. B. HALL AND MICHAEL D. SLOAINE
FIG. 1. MAP OF THE CENTRAL PART OF THE WEST AFRICAN FOREST ZONE, SHOWING KNOWN STATIONS OF TODDALIOIDEAE Symbols for species are: ~lr Araliopsis soyauxii; • Diphasia angolensis; • Oricia suaveolens; • Teclea verdoorniana; • Vepris heterophylla Some of these records are from sample plots recorded by ourselves (J BH and M DS). and some from herbarium records. Broken lines delimit forest types as defined [23]: we--Wet Evergreen; me---Moist Evergreen; ue--Upland Evergreen; ms--Moist Semi-deciduous; ds--Dry Semi-deciduous; sin--Southern Marginal; so--South-east Outlier. National boundaries shown by .--; Volta Lake solid black; savanna hatched. TABLE 1. KEY TO THE GENERA OF WEST AFRICAN MEMBERS OF THE RUTACEAE (TODDALIOIDAE) 1. Large tree to 30 m high with cylindrical trunk; leaflets 5-9; stamens 8; carpels 4(-5). completely fused in fruit; each carpel with 2 ovules, both of which form seeds; pyrenes with adaxial aperture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Araliopsis soyauxii Small understorey trees to 15 m high; leaflets 3: stamens 4(-8): carpels 1-4, fused or free; each carpel with 2 ovules of which only 1 normally forms a seed; pyrenes closed . . . . . . . . . . . . . . . . . . 2 2. I nflorescences glabrous; flowers sessile or with pedicels shorter than calyx; leaflets coriaceous, with strongly decurved margins; ova ry glabrous; carpels 1-2, if two then fused completely; stamens 4-8 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Vepris heterophylla* Inflorescences axes pubescent; flowers with more or tess slender pedicels exceeding calyx; leaflets chartaceous, with more or less flat margins; ovary pubescent; carpels 1-4 not completely fused; stamens always 4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 3. Carpel solitary; leaflets subsessile, glabrous, with tertiary veins more or less parallel to the secondary veins, and areolae elongated in the same direction; male inflorescences 3-8 cm long, with 2-3 orders of branching . . . . . . . . . . . . . . . . . . Teclea verdoomiana Carpels 2-4 with common stigma; leaflets petiolulats, glabrous or pubescent; tertiary nerves with admedial divergent branching, and areolae more or less isodiametric; male inflorescences 10-20 cm long, with 3-4 orders of branching . . . . . . . . . . . . . . . . . . . . . . 4 4. Carpels 4, ovaries free to the base, mature leaflets glabrous Oricia suaveolens Carpels 2, ovaries fused almost to the summit; mature leaflets hairy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Diphasia angolensis * The artificiality of the distinction between the genera Vepris and Teclea is illustrated by V. heterophylla where the stamens may equal the petals and the carpel be solitary (as in Teclea) or the stamens be up to twice this number, and the carpels be two and fused (as in Vepris).
usually twice the number of petals, and endosperm usually present in the seeds. Waterman [8] has discussed the defects of this system of classification from a chemical point of view and has illustrated how the alkaloid content can be employed to construct a putative phylogeny for the African Toddalioideae differing radically from that proposed by Verdoorn [9]. Subsequently it has proved
possible to investigate further the alkaloid chemistry of all five of the species under discussion. The results of those analyses are given in Table 2. With the exception of the quaternary phenylethylamine candicine (15) all of the alkaloids isolated are of either acridone or furano/pyranoquinoline structural types. The inter-relationship between these alkaloids has been dealt with comprehensively in a recent review [17] and is summarised in Scheme 1. The most striking similarity noted in the alkaloid chemistry was that between D. angolensis and T. verdoorniana. Both contain, in a number of samples, the acridone evoxanthine (4) as the major alkaloid with minor amounts of the closely related 1,3-dimethoxyN-methylacridone (2). Two further acridones were identified with certainty, tecleanthine (5) from T. verdoorniana and arborinine (3) from D. angolensis; both were detected by TLC as trace constituents in the alternate species. A further prominent compound in these samples was the amino-benzophenone tecleanone (1) which has been implicated in the biogenesis of acridones [18]. Furoquinolines occurred in only trace amounts in all samples; the commonest alkaloid of this group, skimmianine (8), could be identified in all. The more highly substituted halfordinine (11) was confirmed in T. verdoorniana and inferred, by TLC, in D. angolensis. Tecleanone, together with some of the acridone alkaloids mentioned, have previously
BIOCHEMICAL SYSTEMATICSAND ECOLOGYOF THE TODDALIOIDEAE
241
TABLE 2. ALKALOIDSAND TRITERPENESREPORTEDFROMTHE WESTAFRICANTODDALIOIDEAE ACR
PQL
FOL
PEL
TRT
v v
I
"$ n-
4. s o y a u x i i t t ~. angolensis O, s u a v e o l e n s - - G H O, s u a v e o / e n s - - N I G T. verdoorniana V. heterophyl/a
+
+ +
+" +
+ --
+
+
?
+ + +" +
+
+
+
+
+" +*
+
1
--
+
+
+*
+
--
+
+
--
+
+
+
--
+
+
+
1
+
--
+
+
8,10 8,12 13 14 8,12,15 16
+
--
+
--
+
1
• ACR=Acridones;FOL=Furoquinolines;PQL=Pyranoquinolines;PEL=Phenylethylamines;TRT=Triterpenes ?=probablypresent(tentativeTLC identification); •=reportedherefor first time t A. soyauxii also containstrace amountsof (18) and (19); ~ A sampleof A. soyauxfi from Gabonis reported [11] to contain (7-10, 12, 13 and 20)
OMe
~
~ ~ ' ~ O T I (6) Me| \ ! \
o
JL
(7)
~)
~oHMeO
I/~COQH
\c-T/.8
\eubet.
~.
OMe
\ "~-~C-6/7
\
~
MeO"~
,, O
~
00Me
~J:~" NH23x CZ 0"-~ ~ O M e H + C5Anthronilic NH 'Acridone Precursor' I (I) ~Furo/Pyronoquinolone Acid Me ~ Precursor
~L~L-N>LO>Y-OH ' i Me
z
O OMe
MeO " ~Y"T ~MeO ~J,~,.~L,N~.0 \ (14) M/
,~- -u(e}
OMe
R 2 R I ~
I (13} Me
O-
OMe
/
(2) Me
(12) R~, Rz=O-CH2-O
OMe
OMe
(3) Me
l
°°> 141 Me
111) RI iRzlOMe
OH
0 OMe
(9) RI =R2=OMe (10) RI "R2=O-CH2-O
ONe
O
__~ ~ I T ~ OMe ~/-'~-N ~
o OMo
"~
@o, O
MeO
Me
(5)
SCHEME1. RELATIONSHIPSBETWEENANTHRANILICACID DERIVEDALKALOIDS OF THE WEST AFRICAN TODDALIOIDEAE (N.B. The arrowsare not meanttu implyimmediatebiogeneticrelationshipsbetweenalkaloids)
been reported from Tec/ea grandifo/ia Engl. [19]: the isolations being from material of Ghanaian origin. These reports undoubtedly refer to T. verdoorniana since T. grandifo/ia, although existing as a separate taxon in more southern areas of the rain forest, does not occur in the region under discussion. This confusion has arisen from the erroneous assumption [20] that T. verdoorniana and T. grandifolia were conspecific. The complement of acridone alkaloids in two collections of O. suaveo/ens from Ghana
showed considerable similarity to that of the above species. Again, evoxanthine was the major base present and was accompanied by 1,3-dimethoxy-N-methylacridone and, probably, tecleanthine. Only arborinine could not be detected. Furthermore tecleanone was again present in significant quantity and it is noteworthy that, assuming reports for T. grandifofia do refer to T. verdoorniana, the three species so far discussed represent the only known sources of this compound. The less abundant furoquinoline alkaloids were
242
PETER G. WATERMAN, IBRAHIM A. MESHAL, J. B, HALL A N D MICHAEL D. SWAINE
represented by halfordinine accompanied by, instead of the 7,8-substituted skimmianine, the corresponding 6,7-substituted kokusaginine (9). In complete contrast a Nigerian sample of this species gave a single alkaloid in high concentration which proved to be identical with the previously reported [14] angular pyranoquinoline, oricine (14). However a careful examination of this material did reveal the presence of small quantities of evoxanthine. Whilst it is outwith the area under consideration it should be noted that O. gabonensis Pierre, the only other species of the genus as yet investigated for its alkaloids, is also reported to contain evoxanthine as the major alkaloid [21 :]. Neither of the remaining species could be shown to contain acridones. The major alkaloid of V. heterophylla was found to be, in agreement with previous reports (as T. sudanica) [22:], flindersiamine (12) which was accompanied by smaller amounts of skimmianine and maculine (10). The presence of appreciable quantities of candicine (15), a metabolite of tyrosine rather than anthranilic acid which is common in Zanthoxylum [17], was surprising but has a precedent in the Toddalioideae, in Tec/ea simpficifofia (Engl.) Verdoorn [8]. Ghanaian material of A. soyauxii
I I t I I I I I I I
55 --
ue 50 -
45 -
~
(reported as A. tabouensis) showed the most individual alkaloid spectrum among the five species. In addition to the typical furoqui nolines, skimmianine and halfordinine, two samples of this species contain considerable quantities of the 2-isopropyl-2,3-dihydrofuroquinolines, Nmethylplatydesminium ion (6) and its isofuroquinoline derivative, isoplatydesmine (7). The linear pyranoquinoline ribalinine (13) was also present in significant amounts and traces of the indoloquinazoline alkaloids evodiamine (18) and rhetsinine (19) were established. Material of A. soyauxii from Gabon has recently been reported [11~] to produce a similar range of alkaloids plus an unusual angular furoquinoline araliopsine (20) which may have a close biogenetic relationship to oricine. A further distinction of A. soyauxii was seen in the presence of about 2% of the sterol-derived limonoid precursor flindissol (17) in the bark. This could not be detected in any of the other species. The common pentacyclic triterpene lupeol (16) was a feature in all samples studied. The most striking and surprising observation is that three species (D. angolensis, O. suaveolens--Ghanaian material, and T. verdoorniana) have virtually identical alkaloid corn-
-
""; .':'"
•
ms
•
..:
•
_1
•
\
Sill
\\\
:
•
".
•
:•
a ~
~,
-we
I I
I~i
20
•
%% file
\ ~
I 30
SO
• \ \
~1
I
40
50
\~1 60
Axis I FIG. 2. ORDINATION BASED ON 155 G H A N A I A N FOREST PLOTS A N D PARTITIONED INTO FOREST-TYPES (see [23] for details). Abbreviations for forest-types, and symbols for species, as in Fig. 1. Each symbol marks the position on the ordination diagram of a forest plot in which the corresponding member of Rutaceae (Toddalioidees) was recorded, This position is determined by the mean scores [23] on axes I and VI of ell species present in the plot. Oblique lines indicate plots in Ivory Coast; "'N" indicates the Nigerian plot.
I
7O
BIOCHEMICALSYSTEMATICSAND ECOLOGYOF THE TODDALIOIDEAE
243
ordination method used was "reciprocal averaging" [24], which has the advantage that, for (is) any axis, the stand ordination score is directly proportional to the mean of the ordination scores of the species constituting the stand. The ordination positions (Fig. 2) of all Ivory Coast and Nigerian plots, and many of the Ghana plots, were thus readily obtained using the species ordination scores from the original Ghana ordination, for the species present. It can be seen from Fig. 2 that the three (le) species have, to a great extent, coherent and exclusive ranges on the ordination diagram. This picture is consistent with the hypothesis that a wide-ranging ancestral species has differentiated into three daughter vicariant [~~N OH ~N.~O species of different ecological tolerances. The H 0 "~V N~.M severely limited distribution of both types of (19) i ~ • alkaloid involved, acridones entirely and furoquinolines largely confined to the Rutaceae [17], could be taken as implying a monophyletic evolution for them within the family. As a corollary it would seem likely that, if speciation has occurred, the common ancestor already possessed the ability to synthesise both types. As neither of the two remaining species of Toddalioideae extant within the area seem I capable of synthesising the acridone nucleus it (20) Me does not seem possible that either of them plements (Table 2) although they are placed in could be close to the pro-species. Acridone and furoquinoline alkaloids have different genera. Such close similarity is thought more likely to result from fairly been found in the sympatric species Zanrecent common ancestry than from phyletic thoxylum leprieurii Guill. Et Perr [25] of the convergence. This assumption is supported by subfamily Rutoideae. Whilst this similarity is their close morphological similarity (see Table noteworthy (Z. leprieurii contains additional 1 ) in most characters other than those used for alkaloid types not observed in any of the generic separation e.g. in habit, colour of bark above) and supports proposed inter-relationand slash, leaflet dimension and shape etc. ships between the subfamilies it seems Illustrative of this point are two specimens of impossible, on morphological grounds, to O. suaveolens found recently in the Forest suggest a direct and recent derivation of the Herbarium at Kumasi, Ghana (both with male three acridone producing Toddalioideae from flowers), one of which had been identified as this putatively more primitive species. T. verdoomiana and the other as D. angolensis/ The only Nigerian record for the trio is Ecologically these three species are distinct. notably discrepant; O. suaveolens is known It is apparent from Fig. 1 that O. suaveolens there in only one station, which is Dry Semi(in Ghana and Ivory Coast) favours wetter deciduous forest, whereas in Ghana and forest areas than D. angolensis, while T. Ivory Coast it centres on Wet and Moist verdoorniana is restricted to dry forests near the Evergreen forest. It is therefore of great savanna boundary. Ecological ranges are interest that the Nigerian population should shown more clearly in Fig. 2 which is based on also be chemically anomalous; it may well be a two-dimensional ordination of 155 sample that this population is in process of speciation. plots (each of 0.0625 ha), taken in closed If this is so then speciation has, in this case, canopy forest throughout the Ghana forest produced a much simplified alkaloid pattern, zone; the ordination being partitioned to define presumably through the loss or drastic reforest types [23]. Only plots containing duction of enzymes required for formation of Toddalioideae are shown in Fig. 2. The the acridone nucleus and cyclisation of the HOJ~+N(CH3) 3
244
PETER G. WATERMAN. IBRAHIM A. MESHAL, J. B. HALL AND MICHAEL D. SWAINE
furan ring. This has permitted the formation of large quantities of the thermodynamically preferred angular pyranoquinoline nucleus [26]. The unusual methoxy substitution pattern of the pyranoquinoline oricine is identical to that of the furoquinoline kokusaginine, which was found in the Ghana/an material of Or/c/a, thus indicating retention of the ability to substitute the anthranilate nucleus at C-6 and C-7 without substitution at C-8. Of the remaining species, A. soyauxii has a narrower range than that of O. suaveolens, but the two species are centred on the same part of the ordination (Fig. 2). As they differ markedly in habit and in alkaloid content it is considered unlikely that they are in direct competition. As we have already observed, A. soyauxii has the most individual range of alkaloids of the five species studied. The absence of acridones and the emphasis on simple furoquinoline and pyranoquinoline alkaloids might be taken, as a corollary to the situation with the Nigerian population of O. suaveo/ens, to indicate a relatively advanced phylogenetic position. In that context it is interesting to observe that in Nigeria, where O. suaveolens has moved away from Arafiopsis into a drier habitat it has produced a pyranoquinoline alkaloid similar to that of the latter. It could be argued therefore that where the two species occur together in Ghana the development of similar alkaloid spectra would be detrimental to both species, but that in Nigeria no such limitation exists. The reason for a change in alkaloid patterns in Oricia on moving away from Arafiopsis can only be speculated upon. On present distribution data it would seem very likely that D. ango/ensis into whose niche the Nigerian Oricia population seems to have moved, must have been present in that area at some time in the past. The development of aberrant alkaloid chemistry in that Oricia population could perhaps have resulted initially from avoidance of chemical overlap with a sympatric Diphas/a population. It is also noteworthy that in Gabon where A. soyauxii and O. gabonensis occur together an alkaloid diversity very similar to that seen in Ghana has developed. Occupying the driest end of the forest spectrum is V. heterophy//a (Fig. 2). It differs markedly from T. verdoorniana, the species ecologically closest to it, by producing relatively large amounts of furoquinolines and candicine whilst the latter yields mainly acridones plus traces of furoquinolines.
Conclusions Ashton's [27, 28] pioneering work in Tropical Asia has shown the prevalence of niche separation between members of groups of closely allied sympatric species in the Dipterocarpaceae. This phenomenon has scarcely been explored in Tropical Africa, but the present group of species appear to provide an example. Ashton [28] was often able to suggest possible adaptional significance to morphological differences between his species, The Toddalioideae described in the present paper are, morphologically, very similar; their restriction to particular ranges of forest-type must presumably be due to physiological adapt/on, as yet uninvestigated, The importance of secondary plant products in adapting a species for defence against attack by herbivores is now widely accepted [29, 30]. The range occupied by any species, and the density it achieves there, will be determined not only by the physical parameters of its environment, but also by the cohort of herbivores to which it is susceptible or resistant. Janzen [31] has suggested that two ecologically associated species which have similar chemical constitutions may be subjected to greater pest pressure than would either if they were chemically different. The situation among the West African Toddalioideae supports this hypothesis: the species with similar chemistry (T. verdoorniana, D. ango/ensis and O. suaveo/ens) have different ecological ranges; the species with similar ranges (O. suaveo/ens and A. soyauxii) differ chemically. It may be added in parentheses that the great diversity of alkaloids in these plants has not ensured their competitive success. They display, in fact, the characteristics of an ancient, relict group, the species being mostly rare and showing wide disjunction. Finally we would draw attention to the report [32] of a number of closely related furoquinoline and acridone alkaloids [33, 34] in Acronych/a bauer/Schott. (Toddalioideae), Euodia xanthoxyloides F. Muell. and Melicope fareana F. Muell. (both Rutoideae), three species endemic to the rain forest of Queensland, Australia. At first sight this would appear to indicate an analogous situation to that discussed in the present paper. Experimental Plant mater~a~. Diphasia angolensis (i) Neung Forest Reserve, near T a r k w a - - v o u c h e r
ENTI 4 9 0 A at E; (ii)
BIOCHEMICAL SYSTEMATICSAND ECOLOGYOF THE TODDALIOIDEAE Bosuso--voucher ENTI 490B at E; (i/i) Tarkwa-voucher STR 058 at E; (iv) Begoro District--voucher STR 059 at E. Teclea verdoorniana (i) Awutu-Winneba Road--voucher ENTI 390 at E; (ii) Ioc. cit.--voucher ENTI 789 at E. Vepris heterophylla (i) Shai Hills-voucher J. B. HALL GC 46119 at GC and E. Oricia suaveolens (i) Mamiri Forest Reserve (saplings)voucher D, K. ABBIW GC 46120 at GC and E; (ii) Bronikrom--voucher J. B. HALL and NABOOH GC 46626 at GC and E; (i/i) Cocoa Research Institute of Nigeria (formerly part of Gambari Forest Reserve) Araliopsis soyauxfi (i) Fure Forest Reserve, near Prestea --voucher ENTI 461 at E;/oc. cir.--voucher ENTI 793 at E. Identification of alkaloids and triterpenes. The identification of most of the compounds discussed in this paper has already been detailed in earlier publications [8, 10, 12-1 6]. The following are reported for the first time: (a) from D. ango/ensis: 1,3-dimethoxy-N-methylacridone (2)--isolated from the CHCI3 extracts of all samples by preparative TLC on alumina (eluting with C6HB-EtOAc 1:4); identical in all respects (m.m.p., UV, IR, MS, TLC) with an authentic sample. Tecleanthine (5) and halfordinine (11) were detected as trace constituents only and were tentatively identified by TLC comparison (3 systems) with authentic samples isolated from T. verdoorniana and A. soyauxfi respectively; (b) from O. suaveolens: tecleanthine was tentatively identified, by the same method as above, as a trace constituent in GC 46120. Sample GC 46626 proved, on TLC examination, to be identical to GC 46120. Nigerian material: oricine (14) was isolated and identified in the same manner as previously reported [14]. Preparative TLC of petrol and CHCI3 extract respectively gave lupeol (16) and evoxanthine (4) identical in all respects with these compounds isolated from G hanaian material of this species [13]; (c) from A. soyaux/i: lupeol was identified in the petrol extracts after removal of flindissol. A c k n o w l e d g e m e n t s - - T h e authors wish to thank Mr. A. A. Enti, Forestry Enterprises (Ghana) Ltd., Mr. D. K. Abbiw, Dept. of Botany, University of Ghana, and Dr. John B. Hall, Dept. of Forest Resource Management, University of Ibadan, Nigeria, for their help in the collection of plant material. One of us (I.A.M.) thanks the University of Rhiyad, Saudi Arabia, for the award of a scholarship.
References 1. Engler, A. (1964) Syllabus der Pf/anzenfamilien (Melchior, H., ed.) 12th. Edn., p. 262, Borntrager, Berlin. 2. Engler, A. (1931 ) The Rutaceae, in Die Naturlichen Pf/anzenfami//en (Engler, A. and Prantl, K. eds.) 2nd. Edn., Vol. 19a, p. 187, Engelmann, Leipzig. 3. Keay, R. W. J. (1958) The Rutaceae, in Flora of West Tropical Africa 2nd. Edn., Vol. 1, p. 683, Crown Agents, London.
245
4. Voorhoeve, A. G. (1965) Liberian High Forest Trees, p. 416, Centre for Agricultural Publications and Documentation, Wageningen. 5. Letouzey, R. (1963) Rutaceae, in F/ore du Gabon, Vol. 6, p. 3, Museum National d'Histoire Naturelle, Paris. 6. Letouzey, R. (1963) Rutaceae, in F/ore du Cameroun Vol. 1, p. 39, Museum National d'Histoire Naturelle, Paris. 7. Letouzey. R. (1966) Adansonia 6, 243. 8. Waterman. P. G. (1973) Biochem. System. Et Ecol. 1, 153. 9. Verdoorn, I. C. (1926) Kew Bull., 389, 10. Fish, F., Meshal, I. A. and Waterman, P. G. (1976) Planta Medica 29, 310. 11. Vaquette, J., Pousset, J, L., Hifnawy, M. S., Fournet, A., Bouquet, A. and Cave, A. (1976) Phytochemistry 15, 743. 12. Waterman, P. G. (1975) Phytochemistry 14, 2092. 13. Fish, F., Meshal, I. A. and Waterman, P. G. (1978) P/anta Medica 33, 228. 14. Abe, M. O. (1971 ) Phytochemistry 10, 3328. 1 5. Fish, F., Meshal, I. A. and Waterman. P. G. (1976) J. Pharm. Pharmacol. 28, 72P. 16. Fish, F., Meshal, I. A. and Waterman, P. G. (1977) Fitoterapia 48, 1 70. 17. Waterman, P. G, (1975) Biochem. System. E* Ecol. 3, 149. 18. Khan, M. S., Lewis, J. R. and Watt, R. A. (1975) Chem. Ind., 744. 19. Popp, F. D. and Chakraborty, D. P. (1964) J. Pharm. Sci., 53, 968; Casey, A. C. and Malhotra, A. (1975) Tetrahedron Lett. 401. 20. Irvine, R. F. (1961 ) WoodyP/ants of Ghana, p. 488, Oxford University Press. 21. Vaquette, J., Cave, A., Fournet, A. and Bouquet, A. (1975) P/anta Med. Phytother. 9, 304. 22. Paris, R. R. and Stambouli, A. (1959) Compt. Rend. 248, 3736. 23. Hall, J. B. and Swaine, M. D, (1976) J. Ecol. 64, 913. 24. Hill, M. O. (1973) J. Ecol. 61,237. 25. Fish, F. and Waterman, P. G. (1972) Phytochemistry 11, 3007. 26. Collins, J. F., Gray, G. A., Grundon, M. F., Harrison, D. M, and Spyropoulos, C. G. (1973) J. Chem. Soc. Perkin Trans I, 94. 27. Ashton, P. S. (1964) Oxford For. Mem. 25, 1. 28. Ashton, P. S. (1969) Biol. J. Linn. Soc. 1, 155. 29. Levin, D. A. (1976) Ann. Rev. Ecol. System. 7, 121. 30. Rhoades, D. F. and Cates, R. G. (1976) Rec. Adv. Phytochem. 10, 168. 31. Janzen, D. H. (1973) Chemistry in Evolution and Systematics (Swain, T., ed.) p. 529, Butterworths, London. 32. Hughes. G. K., Lahey, F. N., Price, J. R. and Webb, L. J. (1948) Nature 162, 233. 33. Price, J. R. (1963) Chemical Plant Taxonomy (Swain, T., ed.) p. 429, Academic Press, London. 34. Hegnauer, R. (1973) Chemotaxonomie der Pf/anzen, Vol. 6, p. 174, Birkhauser, Basle.
0305-1978/78/0901--0239502.00/0
Biochemical Systematics and Ecology of the Toddalioideae in the Central Part of the West African Forest Zone* PETER G. WATERMAN and IBRAHIM A. MESHAL and J. B. HALL and MICHAEL D. SWAINE Department of Pharmaceutical Chemistry, University of Strathclyde, Glasgow G1 1XW, Scotland Department of Botany, University of Ghana, Legon, Ghana Key Word I n d e x - - Araliopsis soyauxii: Diphasia angolensis; Oricia suaveolens; Teclea verdoomiana; Vepris heterophylla; Rutaceae;Toddalioideae; alkaloids; triterpenes; chemotaxonomy; chemical ecology. Abstract--The distribution, ecology and secondary metabolism of five species of Rutaceae (subfamily Toddalio-
ideae), Araliopsis soyauxii, Diphasia angolensis, Oricia suaveolens, Teclea verdoomiana, and Vepris heterophylla, occurring within the central part of the West African ForestZone is reviewed. It is demonstratedthat the five species exhibit niche separationin such a way that no two specieswith similar complementsof secondarymetabolitesappear to be in direct competition. It is suggested,in support of Janzen's hypothesis,that this situation may haveevolved to alleviate pest pressure. The chemical variation between Ghanaian and Nigerian populations of O. suaveolens is discussed in the light of observations on their ecology. Possiblephylogenetic relationships between these species are discussed. Introduotion
Results and discussion
The Rutaceae is divided into three major subfamilies, Rutoideae, Toddalioideae and Aurantioideae plus four minor, often disputed taxa [1]. Engler [2] recognised eight genera of Toddalioideae in Tropical Africa. These comprise shrubs and trees characterised by digitate leaves with 3-5 leaflets, small, generally unisexual flowers in paniculate inflorescences, and drupaceous fruits. The genera are distinguished, rather artificially, by the number of stamens (equal to or twice the number of petals), number of carpels (1, 2, or 4-5), number of ovules per carpel (1 or 2), and degree of fusion of fruiting carpels. Keay [3] in his revision of the West African species stated that the limits between these genera needed to be thoroughly revised, and this opinion was endorsed by Voorhoeve [4]. The problem is exemplified by Letouzey [5] who includes in his revision of Rutaceae from Gabon a number of taxa of Toddalioideae which, because of incomplete knowledge of flowers of one sex, or of fruits, cannot be ascribed with certainty to any genus.
Five genera have been recorded in the area considered in the present paper (Ivory Coast eastwards to the 5 ° E. meridian--Fig. 1), represented by five species: Araliopsis soyauxii Engl. (which we consider to include A. tabouensis Aubr6v. ~t Pellegr.) extending from Liberia to Gabon; Diphasia angolensis (Hiern) Verdoorn (synonym D. k/aineana Pierre [6]) from Guinde to Angola; Oricia suaveolens (Engl.) Verdoorn from Guinde to Zafre; Teclea verdoorniana Exell Et Mendonqa from Sierre Leone to Zafre; and Vepris heterophylla (Engl.) Letouzey (previously known as Teclea sudanica A. Chev. [7]) from Ghana to Mali and Cameroun. They can be distinguished by the key given in Table 1. According to Engler's classification [2], Teclea is placed in the subtribe Amyridinae, distinguished by a unilocular ovary; Diphasla and Oricia in the Oriciinae, distinguished by 2 - 4 incompletely connate carpels, stamens usually twice the number of petals, and endosperm usually present in the seed; and Araliopsis and Vepris (together with the other ° Part X in the series "'Chemosystematics in the African genera, Toddalia, Toddaliopsis and Rutaceae". For Part IX see Waterman, P. G., and Gray, Oriciopsis) into the Toddaliinae characterised A. I. (1978) Phytochemistry 17, 845. by 2 - 5 completely connate carpels, stamens (Received for publication 10 January 1978) 239
240
PETER G. WATERMAN, IBRAHIM A. MESHAL. J. B. HALL AND MICHAEL D. SLOAINE
FIG. 1. MAP OF THE CENTRAL PART OF THE WEST AFRICAN FOREST ZONE, SHOWING KNOWN STATIONS OF TODDALIOIDEAE Symbols for species are: ~lr Araliopsis soyauxii; • Diphasia angolensis; • Oricia suaveolens; • Teclea verdoorniana; • Vepris heterophylla Some of these records are from sample plots recorded by ourselves (J BH and M DS). and some from herbarium records. Broken lines delimit forest types as defined [23]: we--Wet Evergreen; me---Moist Evergreen; ue--Upland Evergreen; ms--Moist Semi-deciduous; ds--Dry Semi-deciduous; sin--Southern Marginal; so--South-east Outlier. National boundaries shown by .--; Volta Lake solid black; savanna hatched. TABLE 1. KEY TO THE GENERA OF WEST AFRICAN MEMBERS OF THE RUTACEAE (TODDALIOIDAE) 1. Large tree to 30 m high with cylindrical trunk; leaflets 5-9; stamens 8; carpels 4(-5). completely fused in fruit; each carpel with 2 ovules, both of which form seeds; pyrenes with adaxial aperture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Araliopsis soyauxii Small understorey trees to 15 m high; leaflets 3: stamens 4(-8): carpels 1-4, fused or free; each carpel with 2 ovules of which only 1 normally forms a seed; pyrenes closed . . . . . . . . . . . . . . . . . . 2 2. I nflorescences glabrous; flowers sessile or with pedicels shorter than calyx; leaflets coriaceous, with strongly decurved margins; ova ry glabrous; carpels 1-2, if two then fused completely; stamens 4-8 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Vepris heterophylla* Inflorescences axes pubescent; flowers with more or tess slender pedicels exceeding calyx; leaflets chartaceous, with more or less flat margins; ovary pubescent; carpels 1-4 not completely fused; stamens always 4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 3. Carpel solitary; leaflets subsessile, glabrous, with tertiary veins more or less parallel to the secondary veins, and areolae elongated in the same direction; male inflorescences 3-8 cm long, with 2-3 orders of branching . . . . . . . . . . . . . . . . . . Teclea verdoomiana Carpels 2-4 with common stigma; leaflets petiolulats, glabrous or pubescent; tertiary nerves with admedial divergent branching, and areolae more or less isodiametric; male inflorescences 10-20 cm long, with 3-4 orders of branching . . . . . . . . . . . . . . . . . . . . . . 4 4. Carpels 4, ovaries free to the base, mature leaflets glabrous Oricia suaveolens Carpels 2, ovaries fused almost to the summit; mature leaflets hairy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Diphasia angolensis * The artificiality of the distinction between the genera Vepris and Teclea is illustrated by V. heterophylla where the stamens may equal the petals and the carpel be solitary (as in Teclea) or the stamens be up to twice this number, and the carpels be two and fused (as in Vepris).
usually twice the number of petals, and endosperm usually present in the seeds. Waterman [8] has discussed the defects of this system of classification from a chemical point of view and has illustrated how the alkaloid content can be employed to construct a putative phylogeny for the African Toddalioideae differing radically from that proposed by Verdoorn [9]. Subsequently it has proved
possible to investigate further the alkaloid chemistry of all five of the species under discussion. The results of those analyses are given in Table 2. With the exception of the quaternary phenylethylamine candicine (15) all of the alkaloids isolated are of either acridone or furano/pyranoquinoline structural types. The inter-relationship between these alkaloids has been dealt with comprehensively in a recent review [17] and is summarised in Scheme 1. The most striking similarity noted in the alkaloid chemistry was that between D. angolensis and T. verdoorniana. Both contain, in a number of samples, the acridone evoxanthine (4) as the major alkaloid with minor amounts of the closely related 1,3-dimethoxyN-methylacridone (2). Two further acridones were identified with certainty, tecleanthine (5) from T. verdoorniana and arborinine (3) from D. angolensis; both were detected by TLC as trace constituents in the alternate species. A further prominent compound in these samples was the amino-benzophenone tecleanone (1) which has been implicated in the biogenesis of acridones [18]. Furoquinolines occurred in only trace amounts in all samples; the commonest alkaloid of this group, skimmianine (8), could be identified in all. The more highly substituted halfordinine (11) was confirmed in T. verdoorniana and inferred, by TLC, in D. angolensis. Tecleanone, together with some of the acridone alkaloids mentioned, have previously
BIOCHEMICAL SYSTEMATICSAND ECOLOGYOF THE TODDALIOIDEAE
241
TABLE 2. ALKALOIDSAND TRITERPENESREPORTEDFROMTHE WESTAFRICANTODDALIOIDEAE ACR
PQL
FOL
PEL
TRT
v v
I
"$ n-
4. s o y a u x i i t t ~. angolensis O, s u a v e o l e n s - - G H O, s u a v e o / e n s - - N I G T. verdoorniana V. heterophyl/a
+
+ +
+" +
+ --
+
+
?
+ + +" +
+
+
+
+
+" +*
+
1
--
+
+
+*
+
--
+
+
--
+
+
+
--
+
+
+
1
+
--
+
+
8,10 8,12 13 14 8,12,15 16
+
--
+
--
+
1
• ACR=Acridones;FOL=Furoquinolines;PQL=Pyranoquinolines;PEL=Phenylethylamines;TRT=Triterpenes ?=probablypresent(tentativeTLC identification); •=reportedherefor first time t A. soyauxii also containstrace amountsof (18) and (19); ~ A sampleof A. soyauxfi from Gabonis reported [11] to contain (7-10, 12, 13 and 20)
OMe
~
~ ~ ' ~ O T I (6) Me| \ ! \
o
JL
(7)
~)
~oHMeO
I/~COQH
\c-T/.8
\eubet.
~.
OMe
\ "~-~C-6/7
\
~
MeO"~
,, O
~
00Me
~J:~" NH23x CZ 0"-~ ~ O M e H + C5Anthronilic NH 'Acridone Precursor' I (I) ~Furo/Pyronoquinolone Acid Me ~ Precursor
~L~L-N>LO>Y-OH ' i Me
z
O OMe
MeO " ~Y"T ~MeO ~J,~,.~L,N~.0 \ (14) M/
,~- -u(e}
OMe
R 2 R I ~
I (13} Me
O-
OMe
/
(2) Me
(12) R~, Rz=O-CH2-O
OMe
OMe
(3) Me
l
°°> 141 Me
111) RI iRzlOMe
OH
0 OMe
(9) RI =R2=OMe (10) RI "R2=O-CH2-O
ONe
O
__~ ~ I T ~ OMe ~/-'~-N ~
o OMo
"~
@o, O
MeO
Me
(5)
SCHEME1. RELATIONSHIPSBETWEENANTHRANILICACID DERIVEDALKALOIDS OF THE WEST AFRICAN TODDALIOIDEAE (N.B. The arrowsare not meanttu implyimmediatebiogeneticrelationshipsbetweenalkaloids)
been reported from Tec/ea grandifo/ia Engl. [19]: the isolations being from material of Ghanaian origin. These reports undoubtedly refer to T. verdoorniana since T. grandifo/ia, although existing as a separate taxon in more southern areas of the rain forest, does not occur in the region under discussion. This confusion has arisen from the erroneous assumption [20] that T. verdoorniana and T. grandifolia were conspecific. The complement of acridone alkaloids in two collections of O. suaveo/ens from Ghana
showed considerable similarity to that of the above species. Again, evoxanthine was the major base present and was accompanied by 1,3-dimethoxy-N-methylacridone and, probably, tecleanthine. Only arborinine could not be detected. Furthermore tecleanone was again present in significant quantity and it is noteworthy that, assuming reports for T. grandifofia do refer to T. verdoorniana, the three species so far discussed represent the only known sources of this compound. The less abundant furoquinoline alkaloids were
242
PETER G. WATERMAN, IBRAHIM A. MESHAL, J. B, HALL A N D MICHAEL D. SWAINE
represented by halfordinine accompanied by, instead of the 7,8-substituted skimmianine, the corresponding 6,7-substituted kokusaginine (9). In complete contrast a Nigerian sample of this species gave a single alkaloid in high concentration which proved to be identical with the previously reported [14] angular pyranoquinoline, oricine (14). However a careful examination of this material did reveal the presence of small quantities of evoxanthine. Whilst it is outwith the area under consideration it should be noted that O. gabonensis Pierre, the only other species of the genus as yet investigated for its alkaloids, is also reported to contain evoxanthine as the major alkaloid [21 :]. Neither of the remaining species could be shown to contain acridones. The major alkaloid of V. heterophylla was found to be, in agreement with previous reports (as T. sudanica) [22:], flindersiamine (12) which was accompanied by smaller amounts of skimmianine and maculine (10). The presence of appreciable quantities of candicine (15), a metabolite of tyrosine rather than anthranilic acid which is common in Zanthoxylum [17], was surprising but has a precedent in the Toddalioideae, in Tec/ea simpficifofia (Engl.) Verdoorn [8]. Ghanaian material of A. soyauxii
I I t I I I I I I I
55 --
ue 50 -
45 -
~
(reported as A. tabouensis) showed the most individual alkaloid spectrum among the five species. In addition to the typical furoqui nolines, skimmianine and halfordinine, two samples of this species contain considerable quantities of the 2-isopropyl-2,3-dihydrofuroquinolines, Nmethylplatydesminium ion (6) and its isofuroquinoline derivative, isoplatydesmine (7). The linear pyranoquinoline ribalinine (13) was also present in significant amounts and traces of the indoloquinazoline alkaloids evodiamine (18) and rhetsinine (19) were established. Material of A. soyauxii from Gabon has recently been reported [11~] to produce a similar range of alkaloids plus an unusual angular furoquinoline araliopsine (20) which may have a close biogenetic relationship to oricine. A further distinction of A. soyauxii was seen in the presence of about 2% of the sterol-derived limonoid precursor flindissol (17) in the bark. This could not be detected in any of the other species. The common pentacyclic triterpene lupeol (16) was a feature in all samples studied. The most striking and surprising observation is that three species (D. angolensis, O. suaveolens--Ghanaian material, and T. verdoorniana) have virtually identical alkaloid corn-
-
""; .':'"
•
ms
•
..:
•
_1
•
\
Sill
\\\
:
•
".
•
:•
a ~
~,
-we
I I
I~i
20
•
%% file
\ ~
I 30
SO
• \ \
~1
I
40
50
\~1 60
Axis I FIG. 2. ORDINATION BASED ON 155 G H A N A I A N FOREST PLOTS A N D PARTITIONED INTO FOREST-TYPES (see [23] for details). Abbreviations for forest-types, and symbols for species, as in Fig. 1. Each symbol marks the position on the ordination diagram of a forest plot in which the corresponding member of Rutaceae (Toddalioidees) was recorded, This position is determined by the mean scores [23] on axes I and VI of ell species present in the plot. Oblique lines indicate plots in Ivory Coast; "'N" indicates the Nigerian plot.
I
7O
BIOCHEMICALSYSTEMATICSAND ECOLOGYOF THE TODDALIOIDEAE
243
ordination method used was "reciprocal averaging" [24], which has the advantage that, for (is) any axis, the stand ordination score is directly proportional to the mean of the ordination scores of the species constituting the stand. The ordination positions (Fig. 2) of all Ivory Coast and Nigerian plots, and many of the Ghana plots, were thus readily obtained using the species ordination scores from the original Ghana ordination, for the species present. It can be seen from Fig. 2 that the three (le) species have, to a great extent, coherent and exclusive ranges on the ordination diagram. This picture is consistent with the hypothesis that a wide-ranging ancestral species has differentiated into three daughter vicariant [~~N OH ~N.~O species of different ecological tolerances. The H 0 "~V N~.M severely limited distribution of both types of (19) i ~ • alkaloid involved, acridones entirely and furoquinolines largely confined to the Rutaceae [17], could be taken as implying a monophyletic evolution for them within the family. As a corollary it would seem likely that, if speciation has occurred, the common ancestor already possessed the ability to synthesise both types. As neither of the two remaining species of Toddalioideae extant within the area seem I capable of synthesising the acridone nucleus it (20) Me does not seem possible that either of them plements (Table 2) although they are placed in could be close to the pro-species. Acridone and furoquinoline alkaloids have different genera. Such close similarity is thought more likely to result from fairly been found in the sympatric species Zanrecent common ancestry than from phyletic thoxylum leprieurii Guill. Et Perr [25] of the convergence. This assumption is supported by subfamily Rutoideae. Whilst this similarity is their close morphological similarity (see Table noteworthy (Z. leprieurii contains additional 1 ) in most characters other than those used for alkaloid types not observed in any of the generic separation e.g. in habit, colour of bark above) and supports proposed inter-relationand slash, leaflet dimension and shape etc. ships between the subfamilies it seems Illustrative of this point are two specimens of impossible, on morphological grounds, to O. suaveolens found recently in the Forest suggest a direct and recent derivation of the Herbarium at Kumasi, Ghana (both with male three acridone producing Toddalioideae from flowers), one of which had been identified as this putatively more primitive species. T. verdoomiana and the other as D. angolensis/ The only Nigerian record for the trio is Ecologically these three species are distinct. notably discrepant; O. suaveolens is known It is apparent from Fig. 1 that O. suaveolens there in only one station, which is Dry Semi(in Ghana and Ivory Coast) favours wetter deciduous forest, whereas in Ghana and forest areas than D. angolensis, while T. Ivory Coast it centres on Wet and Moist verdoorniana is restricted to dry forests near the Evergreen forest. It is therefore of great savanna boundary. Ecological ranges are interest that the Nigerian population should shown more clearly in Fig. 2 which is based on also be chemically anomalous; it may well be a two-dimensional ordination of 155 sample that this population is in process of speciation. plots (each of 0.0625 ha), taken in closed If this is so then speciation has, in this case, canopy forest throughout the Ghana forest produced a much simplified alkaloid pattern, zone; the ordination being partitioned to define presumably through the loss or drastic reforest types [23]. Only plots containing duction of enzymes required for formation of Toddalioideae are shown in Fig. 2. The the acridone nucleus and cyclisation of the HOJ~+N(CH3) 3
244
PETER G. WATERMAN. IBRAHIM A. MESHAL, J. B. HALL AND MICHAEL D. SWAINE
furan ring. This has permitted the formation of large quantities of the thermodynamically preferred angular pyranoquinoline nucleus [26]. The unusual methoxy substitution pattern of the pyranoquinoline oricine is identical to that of the furoquinoline kokusaginine, which was found in the Ghana/an material of Or/c/a, thus indicating retention of the ability to substitute the anthranilate nucleus at C-6 and C-7 without substitution at C-8. Of the remaining species, A. soyauxii has a narrower range than that of O. suaveolens, but the two species are centred on the same part of the ordination (Fig. 2). As they differ markedly in habit and in alkaloid content it is considered unlikely that they are in direct competition. As we have already observed, A. soyauxii has the most individual range of alkaloids of the five species studied. The absence of acridones and the emphasis on simple furoquinoline and pyranoquinoline alkaloids might be taken, as a corollary to the situation with the Nigerian population of O. suaveo/ens, to indicate a relatively advanced phylogenetic position. In that context it is interesting to observe that in Nigeria, where O. suaveolens has moved away from Arafiopsis into a drier habitat it has produced a pyranoquinoline alkaloid similar to that of the latter. It could be argued therefore that where the two species occur together in Ghana the development of similar alkaloid spectra would be detrimental to both species, but that in Nigeria no such limitation exists. The reason for a change in alkaloid patterns in Oricia on moving away from Arafiopsis can only be speculated upon. On present distribution data it would seem very likely that D. ango/ensis into whose niche the Nigerian Oricia population seems to have moved, must have been present in that area at some time in the past. The development of aberrant alkaloid chemistry in that Oricia population could perhaps have resulted initially from avoidance of chemical overlap with a sympatric Diphas/a population. It is also noteworthy that in Gabon where A. soyauxii and O. gabonensis occur together an alkaloid diversity very similar to that seen in Ghana has developed. Occupying the driest end of the forest spectrum is V. heterophy//a (Fig. 2). It differs markedly from T. verdoorniana, the species ecologically closest to it, by producing relatively large amounts of furoquinolines and candicine whilst the latter yields mainly acridones plus traces of furoquinolines.
Conclusions Ashton's [27, 28] pioneering work in Tropical Asia has shown the prevalence of niche separation between members of groups of closely allied sympatric species in the Dipterocarpaceae. This phenomenon has scarcely been explored in Tropical Africa, but the present group of species appear to provide an example. Ashton [28] was often able to suggest possible adaptional significance to morphological differences between his species, The Toddalioideae described in the present paper are, morphologically, very similar; their restriction to particular ranges of forest-type must presumably be due to physiological adapt/on, as yet uninvestigated, The importance of secondary plant products in adapting a species for defence against attack by herbivores is now widely accepted [29, 30]. The range occupied by any species, and the density it achieves there, will be determined not only by the physical parameters of its environment, but also by the cohort of herbivores to which it is susceptible or resistant. Janzen [31] has suggested that two ecologically associated species which have similar chemical constitutions may be subjected to greater pest pressure than would either if they were chemically different. The situation among the West African Toddalioideae supports this hypothesis: the species with similar chemistry (T. verdoorniana, D. ango/ensis and O. suaveo/ens) have different ecological ranges; the species with similar ranges (O. suaveo/ens and A. soyauxii) differ chemically. It may be added in parentheses that the great diversity of alkaloids in these plants has not ensured their competitive success. They display, in fact, the characteristics of an ancient, relict group, the species being mostly rare and showing wide disjunction. Finally we would draw attention to the report [32] of a number of closely related furoquinoline and acridone alkaloids [33, 34] in Acronych/a bauer/Schott. (Toddalioideae), Euodia xanthoxyloides F. Muell. and Melicope fareana F. Muell. (both Rutoideae), three species endemic to the rain forest of Queensland, Australia. At first sight this would appear to indicate an analogous situation to that discussed in the present paper. Experimental Plant mater~a~. Diphasia angolensis (i) Neung Forest Reserve, near T a r k w a - - v o u c h e r
ENTI 4 9 0 A at E; (ii)
BIOCHEMICAL SYSTEMATICSAND ECOLOGYOF THE TODDALIOIDEAE Bosuso--voucher ENTI 490B at E; (i/i) Tarkwa-voucher STR 058 at E; (iv) Begoro District--voucher STR 059 at E. Teclea verdoorniana (i) Awutu-Winneba Road--voucher ENTI 390 at E; (ii) Ioc. cit.--voucher ENTI 789 at E. Vepris heterophylla (i) Shai Hills-voucher J. B. HALL GC 46119 at GC and E. Oricia suaveolens (i) Mamiri Forest Reserve (saplings)voucher D, K. ABBIW GC 46120 at GC and E; (ii) Bronikrom--voucher J. B. HALL and NABOOH GC 46626 at GC and E; (i/i) Cocoa Research Institute of Nigeria (formerly part of Gambari Forest Reserve) Araliopsis soyauxfi (i) Fure Forest Reserve, near Prestea --voucher ENTI 461 at E;/oc. cir.--voucher ENTI 793 at E. Identification of alkaloids and triterpenes. The identification of most of the compounds discussed in this paper has already been detailed in earlier publications [8, 10, 12-1 6]. The following are reported for the first time: (a) from D. ango/ensis: 1,3-dimethoxy-N-methylacridone (2)--isolated from the CHCI3 extracts of all samples by preparative TLC on alumina (eluting with C6HB-EtOAc 1:4); identical in all respects (m.m.p., UV, IR, MS, TLC) with an authentic sample. Tecleanthine (5) and halfordinine (11) were detected as trace constituents only and were tentatively identified by TLC comparison (3 systems) with authentic samples isolated from T. verdoorniana and A. soyauxfi respectively; (b) from O. suaveolens: tecleanthine was tentatively identified, by the same method as above, as a trace constituent in GC 46120. Sample GC 46626 proved, on TLC examination, to be identical to GC 46120. Nigerian material: oricine (14) was isolated and identified in the same manner as previously reported [14]. Preparative TLC of petrol and CHCI3 extract respectively gave lupeol (16) and evoxanthine (4) identical in all respects with these compounds isolated from G hanaian material of this species [13]; (c) from A. soyaux/i: lupeol was identified in the petrol extracts after removal of flindissol. A c k n o w l e d g e m e n t s - - T h e authors wish to thank Mr. A. A. Enti, Forestry Enterprises (Ghana) Ltd., Mr. D. K. Abbiw, Dept. of Botany, University of Ghana, and Dr. John B. Hall, Dept. of Forest Resource Management, University of Ibadan, Nigeria, for their help in the collection of plant material. One of us (I.A.M.) thanks the University of Rhiyad, Saudi Arabia, for the award of a scholarship.
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