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Biochemical Systematics and Ecology 31 (2003) 1221–1246www.elsevier.com/locate/biochemsyseco
Phenolics, fibre, alkaloids, saponins, andcyanogenic glycosides in a seasonal cloud
forest in India
S. Mali a, R.M. Borgesb,∗
a National Innovation Foundation, Vastrapur, Ahmedabad 380 015, Indiab Centre for Ecological Sciences, Indian Institute of Science, Bangalore 560 012, India
Received 18 June 2001; accepted 3 February 2003
AbstractWe investigated secondary compounds in ephemeral and non-ephemeral parts of trees and
lianas of a seasonal cloud forest in the Western Ghats of India. We measured astringency,phenolic content, condensed tannins, gallotannins, ellagitannins, and fibre, and also screenedfor alkaloids, saponins and cyanogenic glycosides in 271 plant parts across 33 tree and 10liana species which constituted more than 90% of the tree and liana species of this species-poor forest. Cyanogenic glycosides occurred only in the young leaves ofBridelia retusa(Euphorbiaceae), i.e. in 2.3% of species examined. Alkaloids were absent from petioles, ripefruit and mature seeds examined. Saponins were found in all types of plant parts. Condensedtannins occurred in almost all plant parts examined (93.6%), while hydrolysable tannins wereless ubiquitous (gallotannins in 31.2% of samples, and ellagitannins in 18.9%). Astringencylevels were significantly correlated with total phenolic, condensed tannin, and hydrolysabletannin contents. Condensed tannin and hydrolysable tannin contents were not related. Immatureleaves, flowers, and petioles had high astringency while lower levels were found in fruit.Flowers and fruit had the lowest fibre levels. There was no relationship between relative domi-nance of a species in the forest and the fibre or phenolic contents of its mature leaves. In eachplant part category, the frequency of species containing tannins together with alkaloids orsaponins was significantly lower than the frequency of species containing tannins alone. Therewas, however, no segregation between alkaloids and saponins. 2003 Published by Elsevier Science Ltd.Keywords: Condensed tannins; Hydrolysable tannins; Plant defences; Plant secondary compounds;Ratufaindica; Western Ghats
∗ Corresponding author. Tel.:+91-80-3602972; fax:+91-80-3601428.E-mail address: [email protected] (R.M. Borges).
0305-1978/03/$ - see front matter 2003 Published by Elsevier Science Ltd.doi:10.1016/S0305-1978(03)00079-6
1222 S. Mali, R.M. Borges / Biochemical Systematics and Ecology 31 (2003) 1221–1246
1. Introduction
Data on community-level distribution of secondary compounds (mainly phenolicsand alkaloids) are available for only a few tropical forests in Africa and Asia (McKeyet al., 1978; Gartlan et al., 1980; McKey et al., 1981; Davies et al., 1988; Watermanet al., 1988; Kool, 1992). Especially with regard to phenolics, most of these studieshave focused on only a few estimation methods. For example, most studies have notexamined hydrolysable tannins or the relationship between condensed and hydrolys-able tannins in plant parts. In this paper, we report on a quantitative analysis ofphenolics, condensed tannins, hydrolysable tannins (gallotannins and ellagitannins),bovine serum albumin (BSA) assays for tannin astringency, digestibility reducerssuch as acid detergent fibre (ADF), neutral detergent fibre (NDF) and acid detergentlignin (ADL) for 271 ephemeral and non-ephemeral parts of 33 tree and 10 lianaspecies within a seasonal cloud forest community in the Western Ghats of India. Wealso provide qualitative data on saponins, alkaloids, and cyanogenic glycosides forthese resources. The sampled tree and liana species constituted more than 90% ofthe species at the site, and the samples were collected as part of a larger study onthe foraging strategy of the Malabar giant squirrel Ratufa indica. We restricted ouranalysis to those compounds that have been found to affect the foraging strategy ofarboreal mammalian herbivores such as primates (e.g. Oates et al., 1980; McKey etal., 1981; Waterman and Choo, 1981; Waterman and Kool, 1994) and giant squirrels(Borges, 1989; Borges, 1992). Data on the macro- and micro-nutrients within theseplant parts will be presented elsewhere. Because of the enormous structural diversityand lack of general techniques, we restricted our analysis to the qualitative analysisof toxins. Owing to the structural diversity of tannins and the procedural difficultiesinvolved in their quantitative analysis (Martin and Martin, 1982; Mole and Water-man, 1987a,b; Mole et al., 1989; Waterman and Mole, 1989), we employed onlystandard and improved methods recommended by Waterman and Mole (1994). Wealso used a combination of chemical and protein-precipitating methods to determinethe biological activity of tannins. Therefore, our results are comparable with studiesof other forest communities done elsewhere.
Despite the limitations of our data set, in terms of missing analyses for somemetabolites in some plant parts, owing to factors such as lack of adequate sample,insignificance in the giant squirrel diet or other logistic constraints, we also attemptin this paper to examine the co-occurrence of metabolites such as tannins, alkaloidsand saponins in various plant parts in order to examine predictions about the possiblesynergisms or negative interactions between these metabolites. For example, sincealkaloids and tannins react to form insoluble alkaloid-tannates in herbivore guts pre-venting reactions between tannins and proteins (Freeland and Janzen, 1974), andbecause the surfactant properties of saponins negate the anti-digestibility effects oftannins (Martin and Martin, 1984; Freeland et al., 1985), tannins are expected notto co-occur with either alkaloids or saponins in plant parts. Since alkaloids and sap-onins may have synergistic effects (e.g. Kerharo and Adam, 1974), they may beexpected to co-occur to enhance herbivore deterrence.
In this paper we therefore primarily report on the distribution of secondary com-
1223S. Mali, R.M. Borges / Biochemical Systematics and Ecology 31 (2003) 1221–1246
pounds in various plant parts and secondarily attempt to test a few predictions relat-ing to the co-occurrence of tannins, alkaloids and saponins in these resources.
2. Materials and methods
2.1. Study site
The study area was within the Bhimashankar Wildlife Sanctuary in MaharashtraState, India (19°21�–19°11�N, 73°31�–73°37�E, altitude 900 m, annual precipitation3000 mm). This is a species-poor forest where only eight tree species contribute to85.4% of relative dominance values, and the three most common species (Mangiferaindica [Anacardiaceae], Memecylon umbellatum [Melastomataceae] and Olea dioica[Oleaceae]) contribute to 64.1% of relative dominance (Table 1). The forest is highlyfragmented; our study site was situated in the largest and best protected fragmentconstituting a temple sacred grove (see Borges, 1990, 1993 for further descriptions).
2.2. Sample collection and processing
Samples represented 25 families and 13 orders of plants (Table 1), and thuscovered a wide spectrum of species within the cloud forest. All analysed specieswith only two exceptions (Terminalia bellerica and Terminalia chebula) were partof the natural evergreen community of the cloud forest. As these chemical analyseswere performed as part of a larger study on the foraging ecology of the giant squirrelR. indica (Mali, 1998), our sampling and choice of chemical analyses were designedto understand food preference and food avoidance, and were also influenced by theavailability of adequate sample and other logistic constraints. Samples of bothephemeral and non-ephemeral items were collected at the time of year when theyfeatured most in the diet of the giant squirrel (to control for seasonal variation inphytochemistry, if any) and were dried at 40–50 °C in the field in kerosene ovens.
2.3. Qualitative field tests on fresh material for alkaloids, saponins andcyanogenic glycosides
We field-tested for alkaloids using Dragendorff’s and Mayer’s reagents, and laterperformed confirmatory tests on dried material (Gartlan et al., 1980). For saponindetection, we vigorously agitated small aqueous extracts with distilled water andtook a substantial and long-lasting lather formation to indicate the presence of sap-onins (Trease and Evans, 1972). Some seeds and fruit pulp were difficult to hom-ogenise adequately for saponin extraction and remained untested. We used the picratetest to detect cyanogenic glycosides (Conn, 1979).
2.4. Quantitative phytochemical analysis
The results of the chemical analyses presented here are single values estimatedfrom samples pooled across several plant individuals (Appendix A, Table A1).
1224 S. Mali, R.M. Borges / Biochemical Systematics and Ecology 31 (2003) 1221–1246
Table 1Orders, families and species of trees and lianas for which phytochemical data were obtained and relativedominance of trees in the study site
Order Family Species Relative dominance
TreesCelastrales Celastraceae Cassine paniculata (Wt. and 1.4
Arn.)Cassine sp. –a
Maytenus rothiana (Walp.) –Lobreau-Cal.
Ericales Ebenaceae Diospyros montana Roxb. –Diospyros sylvatica Roxb. 1.5
Sapotaceae Vangueira spinosa Roxb. –Xantolis tomentosa (Roxb.) Raf. 7.0
Symplocaceae Symplocos beddomei Clarke –Gentianales Rubiaceae Bridelia retusa (L.) Sprengel 0.2
Canthium dicoccum (Gaert.) T. –and B.b
Randia dumetorum (Retz.) Poirb 0.04Lamiales Oleaceae Olea dioica Roxb.b 14.3Laurales Lauraceae Actinodaphne angustifolia 0.2
(Blume)Litsea stocksii Hook. 1.0
Malphigiales Clusiaceae Garcinia talbotii Raizada ex. 2.1Sant.
Euphorbiaceae Macaranga peltata (Roxb.). 0.1Muell.-Arg.Mallotus philippensis (Lam.) 1.4Muell.-Arg.
Salicaceae Flacourtia indica (Burman) 0.05Merrill
Myrtales Combretaceae Terminalia bellerica (Gaert.) –Roxb.Terminalia chebula Retz. 0.04
Melastomataceae Memecylon umbellatum N. 15.5Burman
Myrtaceae Syzygium cumini (L.) Skeels 6.5Syzygium gardneri Thw. 2.5
Rosales Moraceae Artocarpus heterophyllus Lam. 0.02Ficus callosa Willd. 3.1Ficus racemosa L. 0.5Ficus religiosa L. 0.09Ficus tsjahela Burman –
Sapindales Anacardiaceae Mangifera indica L. 34.3Meliaceae Amoora lawii (Wt.) Bedd. 1.8
Dysoxylum binectariferum 0.4(Roxb.) Bedd.
Rutaceae Atalantia racemosa Wt. and Arn. 0.4Sapindaceae Lepisanthes tetraphylla (Vahl) 1.7
Radlk.LianasEricales Myrsinaceae Embelia ribes Burman
(continued on next page)
1225S. Mali, R.M. Borges / Biochemical Systematics and Ecology 31 (2003) 1221–1246
Table 1 (continued)
Order Family Species Relative dominance
Fabales Fabaceae Acacia concinna (Willd.). DC.Acacia sp.Mezoneuron cucullatum (Roxb.)Wt. and Arn.
Gentianales Rubiaceae Randia rugulosa (Thw.) Hk.b
Gnetales Gnetaceae Gnetum ula Brongn.Oxalidales Connaraceae Rourea santaloides Dalz. and
Gibs.Ranunculales Menispermaceae Diploclisia glaucescens (Blume)
Diels.Rosales Elaeagnaceae Elaeagnus conferta Roxb.
Rhamnaceae Ventilago bombaiensis Dalz.
a In the relative dominance column, – indicates that the species occurred in such small numbers in thestudy plot that dominance values were below 0.001. Dominance values were not obtained for lianas.
b Species authority citation from Saldanha and Nicolson (1976); for the rest read as Saldanha (1984,1986).
Owing to the large number of different plant resources involved, it was not possibleto examine seasonal variation, if any, in the chemistry of the resources. We estimatedtotal phenolic content by the Folin–Ciocalteu method (modified by Singleton andRossi, 1965; detailed in Waterman and Mole, 1994) using extracts in 50% aqueousmethanol (Martin and Martin, 1982) and tannic acid to construct the standard curve.We used the proanthocyanidin method to estimate condensed tannins (Porter et al.,1986; detailed in Waterman and Mole, 1994) using extracts in 50% aqueous methanol(Martin and Martin, 1982) and quebracho tannin (supplied by Anne Hagerman,Miami University) to construct the standard curve. For hydrolysable tannins, weprepared the sample extracts in 70% acetone. We used the rhodanine method forgallotannins (Inoue and Hagerman, 1988), and constructed the standard curve usinggallic acid. For ellagitannins we used the method of Wilson and Hagerman (1990),and constructed the standard curve using ellagic acid. We estimated the astringencyof tannins using the BSA assay (Hagerman and Butler, 1978; Asquith and Butler,1985) with Remazol brilliant blue and used tannic acid to construct the standardcurve. We determined fibre content (ADF, NDF and ADL) according to Goeringand van Soest (1970).
2.5. Independence of data points
In our analysis of patterns, we have treated each species as an independent datapoint and we have not conducted phylogenetically independent contrasts (PIC). Thisis because our data are from 42 angiosperm and one gymnosperm species within 25families and 13 orders (Table 1; mean number of species per family = 1.75 ±1.06 SD; mode = 1; mean number of genera per family = 1.36 ± 0.57 SD). Therefore,
1226 S. Mali, R.M. Borges / Biochemical Systematics and Ecology 31 (2003) 1221–1246
in almost all cases, we were examining only one species per genus, and one genusper family.
3. Results
3.1. Occurrence of alkaloids, saponins, cyanogenic glycosides and phenolics
Cyanogenic glycosides were not found in any of the plant tissues examined (N= 54 bark items, N = 108 reproductive parts, N = 109 leaf items) except for theflush leaves of B. retusa (Euphorbiaceae). Therefore, only one out of 43 plant species(2.3%) exhibited cyanogenesis. Summaries of the species-wise occurrence of thedifferent compounds in the various plant parts are given in Table 2. Alkaloids werenot found in petioles, semi-ripe and ripe fruit, and mature seeds examined (AppendixA, Table A1). Saponins were found in all types of plant parts (Appendix A, TableA1). Condensed tannins were found in all immature leaves, as well as all petiolesand flowers examined. Hydrolysable tannins were found in 68% of species, and 38%of plant samples. Gallotannins were found in only 31% of the samples examinedwhile ellagitannins were found in still fewer samples (only 19%). Fewer plant speciescontained condensed or hydrolysable tannins in their bark and stems than in otherplant parts.
3.2. Relative concentrations of secondary compounds in plant parts
Immature leaves, flowers, and petioles had high astringency while lower levelswere found in fruit (Table 3). Tree twigs had low levels of astringency, condensedand hydrolysable tannins but high levels of fibre (Table 3). Inner bark had astringencyand condensed tannin levels comparable to that of mature leaves while fibre levelswere lower than those found in twigs (Table 3). Flowers and fruit had low fibrelevels (Table 3). After Bonferroni’s correction (P � 0.001), only immature leaveswere found to have significantly higher astringency, gallotannin and ellagitannin con-tent than tree twigs, flowers were found to have significantly higher gallotannin levelsthan immature leaves, and tree twigs were found to have significantly higher ADFlevels than mature seeds.
3.3. The quantitative relationship between various measures of phenolics
Astringency levels were strongly positively correlated with total phenolic, con-densed tannin, gallotannin and ellagitanin contents (Table 4). Condensed tanninvalues were not correlated with gallotannin or ellagitannin contents. Gallotannin andellagitannin contents were strongly positively correlated with each other (Table 4).Of the fibre components, since ADF, NDF and ADL are all highly correlated witheach other, we used only ADF values in the correlations. We found no significantrelation between any measure of phenolics and fibre after Bonferroni’s correction,except for the negative relationship between gallotannins and ADF (Table 4).
1227S. Mali, R.M. Borges / Biochemical Systematics and Ecology 31 (2003) 1221–1246
Tab
le2
Perc
ent
spec
ies
cont
aini
ngal
kalo
ids,
sapo
nins
and
phen
olic
sin
vari
ous
plan
tpa
rtca
tego
ries
Cat
egor
yA
lkal
oids
Sapo
nins
Ast
ring
ency
Tot
alC
onde
nsed
Hyd
roly
sabl
eG
allo
tann
ins
Ella
gita
nnin
sph
enol
ics
tann
ins
tann
ins
Imm
atur
ele
aves
Tre
es15
.4(1
3)54
.5(1
1)10
0(1
3)10
0(1
2)10
0(1
2)61
.5(1
3)38
.5(1
3)41
.7(1
2)T
rees
and
liana
s23
.5(1
7)53
.3(1
5)10
0(1
7)10
0(1
6)10
0(1
6)58
.8(1
7)37
.3(1
7)43
.7(1
6)M
atur
ele
aves
Tre
es15
.4(2
6)30
.0(2
0)91
.7(1
2)10
0(1
1)72
.7(1
1)54
.5(1
1)36
.4(1
1)37
.3(1
6)L
iana
s14
.3(7
)20
.0(5
)10
0(5
)10
0(5
)10
0(5
)50
.0(6
)50
.0(6
)33
.3(6
)T
rees
and
liana
s15
.1(3
3)28
.0(2
5)94
.2(1
7)10
0(1
7)81
.2(1
6)56
.2(1
7)41
.8(1
7)29
.4(1
7)Pe
tiole
s0
(11)
60.0
(5)
100
(9)
100
(10)
100
(10)
50.0
(10)
50.0
(10)
20.0
(10)
Flow
ers
7.1
(14)
33.3
(9)
100
(14)
100
(13)
100
(13)
50.0
(12)
50.0
(12)
16.7
(12)
Sem
i-ri
pefr
uit
0(7
)0
(1)
83.3
(6)
83.3
(6)
66.7
(6)
33.3
(6)
16.7
(6)
16.7
(6)
pulp
Rip
efr
uit
pulp
0(1
5)42
.8(7
)10
0(1
4)10
0(1
5)93
.3(1
5)20
.0(1
5)20
.0(1
5)6.
7(1
5)M
atur
ese
eds
0(2
1)33
.3(9
)85
.7(2
1)88
.9(1
8)72
.2(1
8)31
.2(1
6)31
.2(1
6)12
.5(1
6)T
ree
twig
s30
.0(1
0)11
.1(9
)90
.0(1
0)10
0(8
)75
.0(8
)0
(5)
0(5
)0
(5)
Inne
rba
rk15
.0(2
0)43
.7(1
6)82
.3(1
7)10
0(1
6)75
.0(1
6)14
.3(1
4)14
.3(1
4)7.
1(1
4)A
llpo
oled
10.8
(148
)36
.5(9
6)93
.6(1
25)
98.3
(118
)86
.3(1
17)
37.5
(112
)31
.2(1
12)
18.9
(111
)
Val
ues
are
expr
esse
das
perc
ent
spec
ies
exam
ined
that
cont
aine
dth
eco
mpo
und
exce
ptfo
rth
e‘A
llpo
oled
’ca
tego
ryw
here
they
indi
cate
perc
ent
sam
ples
anal
ysed
acro
sssp
ecie
s.V
alue
sin
pare
nthe
ses
are
num
ber
ofsp
ecie
sex
amin
edex
cept
for
the
‘All
pool
ed’
cate
gory
whe
reth
eyin
dica
teto
tal
num
ber
ofsa
mpl
esan
alys
edac
ross
spec
ies.
Unl
ess
spec
ified
,va
lues
are
pool
edfo
rtr
ees
and
liana
s.
1228 S. Mali, R.M. Borges / Biochemical Systematics and Ecology 31 (2003) 1221–1246
Tab
le3
Lev
els
ofas
trin
genc
y,to
tal
phen
olic
s,co
nden
sed
tann
ins,
gallo
tann
ins,
ella
gita
nnin
san
dA
DF
inpl
ant
part
s(m
ean
±SD
)
Cat
egor
yA
stri
ngen
cyT
otal
phen
olic
sC
onde
nsed
tann
ins
Gal
lota
nnin
sE
llagi
tann
ins
AD
F
Imm
atur
e11
.37
±10
.54
(17)
2.70
±3.
59(1
6)16
.85
±19
.02
(16)
0.56
±1.
00(1
7)0.
24±
0.38
(16)
33.9
8±
15.9
6(1
7)le
aves
Mat
ure
7.56
±7.
31(1
7)1.
05±
0.74
(16)
13.1
4±
20.2
9(1
6)0.
28±
0.65
(16)
0.05
±0.
14(2
0)33
.18
±10
.12
(17)
leav
esPe
tiole
s10
.42
±7.
52(9
)3.
01±
4.19
(10)
20.3
7±
20.5
7(1
0)0.
10±
0.16
(10)
0.09
±0.
21(1
0)35
.27
±2.
35(9
)Fl
ower
s9.
51±
7.30
(14)
1.99
±2.
04(1
3)18
.82
±21
.56
(13)
2.70
±5.
34(1
3)0.
22±
0.53
(13)
25.9
3±
12.3
9(1
3)R
ipe
frui
t4.
57±
5.43
(14)
1.13
±1.
58(1
5)6.
54±
7.76
(15)
0.05
±0.
14(1
5)0.
01±
0.04
(16)
27.0
0±
14.3
0(1
4)pu
lpM
atur
e4.
13±
6.41
(21)
1.00
±1.
25(1
8)8.
78±
20.4
9(1
8)0.
72±
1.58
(18)
0.05
±0.
13(1
7)16
.10
±11
.90
(21)
seed
sT
ree
twig
s1.
95±
2.77
(10)
0.29
±0.
23(8
)3.
18±
4.34
(8)
0.00
1±
0.00
1(5
)0.
001
±0.
001
(8)
49.7
0±
20.6
7(1
0)In
ner
bark
6.35
±7.
11(1
7)0.
87±
0.76
(16)
16.6
1±
20.6
5(1
5)0.
06±
0.19
(16)
0.03
±0.
09(1
6)38
.48
±14
.07
(17)
Val
ues
are
pool
edfo
rtr
ees
and
liana
s.A
stri
ngen
cyan
dto
tal
phen
olic
sex
pres
sed
aspe
rcen
tta
nnic
acid
,co
nden
sed
tann
ins
aspe
rcen
tqu
ebra
cho
tann
in,
gallo
tann
ins
aspe
rcen
tga
llic
acid
and
ella
gita
nnin
sas
perc
ent
ella
gic
acid
inte
rms
ofdr
yw
eigh
t.V
alue
sin
pare
nthe
ses
are
num
ber
ofsp
ecie
s(N
).
1229S. Mali, R.M. Borges / Biochemical Systematics and Ecology 31 (2003) 1221–1246
Table 4Correlates of various measures of phenolic compounds and fibre
Total Condensed Gallotannin Ellagitannin ADFphenolics tannin
Astringency 0.48 (119)∗∗∗ 0.37 (118)∗∗∗ 0.20 (116)∗∗ 0.22 (115)∗∗ 0.13 (129)∗Total phenolics 0.28 (121)∗∗∗ 0.36 (116)∗∗∗ 0.17 (114)∗ �0.08 (119)Condensed tannin 0.14 (115) 0.02 (113) 0.13 (118)∗Gallotannin 0.32 (113)∗∗∗ �0.2 (116)∗∗Ellagitannin �0.02 (114)
Values are Kendall’s correlation coefficients. Values in parentheses are sample sizes (N). ∗P � 0.05;∗∗P � 0.01; ∗∗∗P � 0.001 (∗∗ is significant after Bonferroni’s correction for multiple tests).
Although correlations were performed between these phenolic measures separatelyfor each plant part type, e.g. mature leaves, the results were not significant afterBonferroni’s correction for multiple tests, except for the positive correlations betweenastringency and total phenolics in mature leaves of trees (Kendall’s t = 0.4, N =17, P � 0.01) and between astringency and total phenolics (Kendall’s t = 0.61 N= 16, P � 0.01), as well as total phenolics and condensed tannins (Kendall’s t =0.58, N = 16, P � 0.01) in inner bark.
3.4. Co-occurrence of condensed and hydrolysable tannins
Since almost all plant parts contained condensed tannins (Table 2), we were unableto examine the segregation between hydrolysable and condensed tannins in plantparts. Within each plant part we, therefore, compared the frequency of species con-taining both hydrolysable and condensed tannins to those containing condensed tan-nins alone using binomial probabilities, and we found that only in ripe fruit pulpwas there a significantly higher frequency of samples that contained condensed tan-nins but also did not contain hydrolysable tannins (N = 15, P � 0.02). Since therewere samples that did not contain gallotannins, we examined the independence ofoccurrence of gallotannins and ellagitannins using a 2 × 2 contingency test, withYates’ correction, and found that there was no segregation between gallotannins andellagitannins in any plant part.
3.5. Co-occurrence of alkaloids, saponins and phenolics
Since tannins occurred in almost all plant parts examined, we were unable toexamine whether the occurrence of tannins and alkaloids or tannins and saponinswere independent of each other. We, therefore, examined whether significantly fewernumbers of plant parts of the different species contained both tannins and alkaloidsor both tannins and saponins than those that contained tannins alone. We did thisfor each plant part category by calculating exact probabilities of the binomial since
1230 S. Mali, R.M. Borges / Biochemical Systematics and Ecology 31 (2003) 1221–1246
our sample sizes for each plant part category were less than 25 (Sokal and Rohlf,1981, 708 pp.). Since all samples that gave positive results for phenolics with theFolin–Ciocalteu reagent were also astringent, and since astringency was measuredfor a greater number of samples (astringency is biologically more relevant to squirrelforaging as it is a measure of protein precipitation by tannins), we used astringencyas an indication of the presence of phenolics in the samples. Except for tree twigs(N = 10) for which non-significant results were obtained, there were significantlymore species that contained only tannins compared to those that contained both alka-loids and tannins in their different parts. For immature leaves: N = 17 species, P� 0.02; mature leaves: N = 18, P � 0.001; petiole: N = 9, P � 0.002; flowers: N= 14, P � 0.001; ripe fruit pulp: N = 14, P � 0.0001; mature seeds: N = 21, P� 0.0001; inner bark: N = 17, P � 0.02. However, with the exception of matureleaves (N = 16, P � 0.002) and tree twigs (N = 9, P � 0.05), the number of speciescontaining both saponins and phenolics in all other categories was not significantlydifferent from those containing phenolics alone. Since there were samples that didnot contain alkaloids, we used a 2 × 2 contingency test, with Yates’ correction, toexamine the pattern of co-occurrence of alkaloids and saponins and found no signifi-cant pattern of segregation between them. Furthermore, many samples containedneither alkaloids nor saponins (Table 2; Appendix A, Table A1).
3.6. Tree dominance and secondary metabolites
We examined the relationship between the relative dominance of tree species andthe fibre and phenolic contents of their mature leaves, since it may be expected thatdominant trees may have higher values of these compounds owing to their higherapparency (sensu Rhoades and Cates, 1976). However we did not find any significantrelationship (Kendall’s correlation coefficients, P � 0.05).
4. Discussion
4.1. Distribution and content of secondary compounds in plant parts
4.1.1. Phenolics and fibreAlmost all plant species in each plant part category we examined contained con-
densed tannins, while hydrolysable tannins were present in as few as 0% (tree twigs)to 61% (immature leaves of trees) of the species in each category. Condensed tanninsare phylogenetically ancient secondary compounds while hydrolysable tannins arelargely restricted to the dicots and are of more recent origin (Kubitzki and Gottlieb,1984; Gottlieb et al., 1995). The lack of a strong detrimental effect of condensedtannins on insect and mammalian herbivores (Waterman and Kool, 1994; Ayres etal., 1997) has led to the belief that condensed tannins have evolved primarily indefence against microbes and fungi owing to their anti-microbial and fungistaticeffects (Azaizeh et al., 1990). In the seasonal cloud forest of Bhimashankar, densecloud settles on the stunted forest canopies, without lifting, for four monsoon months
1231S. Mali, R.M. Borges / Biochemical Systematics and Ecology 31 (2003) 1221–1246
each year (June through September/October). The need for protection against fungiat this time appears to be high and this may explain the ubiquitous presence oftannins in leaves, which is further evidenced by the low leaf litter decay (R.M.Borges, personal observation). The poor, acidic, leached soils (data from IndianBureau of Soil Sciences) and the high levels of insolation at this site may havefurther resulted in phenolics such as condensed tannins being laid down in leavesand even in other plant parts due to overflow (Haslam, 1985) or a pluralistic combi-nation of the various resource-related defence hypotheses (Berenbaum, 1995).
Owing to the dearth of work on hydrolysable tannins in tropical rainforests weare unable to compare our results with other studies but hope that our results willbe useful for further comparisons. Despite their powerful free-radical scavengingactivity and alleged anti-carcinogenic effects (e.g. Sawa et al., 1999), virtually noinformation is available on the effect of hydrolysable tannins on various types ofherbivores in natural systems (but see Whitten and Whitten, 1987; Clifford and Scal-bert, 2000) although their effect on large arboreal herbivores like the giant squirrelR. indica has been investigated (Borges and Mali, in preparation).
Since we have used the Folin–Ciocalteu method for estimating total phenolics,which is recommended by Waterman and Mole (1994) as being better than the earlierFolin–Denis assay, and since all the earlier studies on community-wide distributionof secondary compounds in tropical forests have used the Folin–Denis method (e.g.Gartlan et al., 1980), our levels of total phenolics cannot be compared with otherstudies. However, as we have used the widely applied proanthocyanidin method forcondensed tannins, our condensed tannin levels can be compared (Table 5) and werefound to be nearly identical with the values found for another evergreen forest atKakachi in southern India (Oates et al., 1980) despite the complete non-overlap ofspecies between Bhimashankar and Kakachi. Furthermore, these condensed tanninlevels were also found to be close to the values found for the two African andthe two south-east Asian forests that have been most extensively studied (Table 5).Interestingly, the fibre levels (ADF) of the mature trees at Bhimashankar were alsofound to be nearly the same as those measured at Kakachi (Table 5).
Immature leaves, flowers, and petioles had high astringency while lower levelswere found in fruit. It is possible that either immature leaves, flowers and petiolesactually do have greater protection by biologically active tannins as measured bytheir astringency or that the extractability of phenolics is greater in these tissues.High gallotannin levels were also found by Ossipov et al. (1997) in immature leaves;these levels declined as the leaves matured. Low astringency may be present in fruitas it is known that astringency levels decrease as fruit ripen (Goldstein and Swain,1963). We cannot compare astringency levels between various stages of the samefruit owing to lack of sample sizes. Tree twigs had the lowest levels of astringency,condensed and hydrolysable tannins but the highest levels of fibre. Tree twigs haverarely been analysed chemically (Waterman and Kool, 1994). This pattern of allo-cation of secondary compounds to tree twigs may be a general strategy as twigs areprotected by high lignin levels and therefore do not require protection from othercompounds. Inner bark had astringency levels and condensed tannin levels compara-ble to those of mature leaves while fibre levels were lower than those of twigs. As
1232 S. Mali, R.M. Borges / Biochemical Systematics and Ecology 31 (2003) 1221–1246
Tab
le5
Com
pari
son
ofB
him
asha
nkar
with
othe
rtr
opic
alfo
rest
s
Para
met
erB
him
asha
nkar
,In
dia
Kak
achi
,In
dia
Kib
ale,
Uga
nda
Dou
ala-
Ede
a,Se
pilo
k,B
orne
oK
uala
Lom
pat,
Cam
eroo
nM
alay
sia
Alti
tude
(m)
910
1325
1400
Low
land
�20
0L
owla
nd�
200
Low
land
�20
0R
ainf
all
(mm
)30
0030
8014
85N
A30
0020
00Fo
rest
type
Sem
i-ev
ergr
een
Eve
rgre
enE
verg
reen
Eve
rgre
enE
verg
reen
Eve
rgre
enA
DF
(fibr
e)a
35.1
9(1
9.0–
47.3
7)39
.4(2
4.0–
55.1
)35
.4(1
0.3–
67.8
)47
.0(2
0.8–
77.2
)58
.3(4
0.7–
71.7
)46
.1(2
1.5–
73.2
)C
onde
nsed
tann
inin
mat
ure
6.86
(0–2
3.40
)6.
9(0
–22.
0)5.
8(0
–39.
6)5.
4(0
–17.
0)8.
8(0
–37.
0)4.
8(0
–30.
5)le
aves
a
N15
1423
3817
33
AD
F,ac
idde
terg
ent
fibre
;N
,nu
mbe
rof
spec
ies;
NA
,ra
infa
llva
lue
unav
aila
ble
for
Dou
ala-
Ede
afr
omab
ove-
men
tione
dso
urce
s.N
umbe
rsin
pare
nthe
ses
indi
cate
rang
eof
valu
es.
aV
alue
sfo
rA
DF
and
cond
ense
dta
nnin
are
mea
npe
rcen
tson
dry
wei
ght
basi
s(v
alue
sot
her
than
for
Bhi
mas
hank
arar
eob
tain
edfr
omG
artla
net
al.,
1980
;O
ates
etal
.,19
80;
Wat
erm
anet
al.,
1988
).
1233S. Mali, R.M. Borges / Biochemical Systematics and Ecology 31 (2003) 1221–1246
indicated by Milton (1979) and Waterman (1984), we also found that the secondarychemistry of flowers is more comparable to foliage that any other plant part.
4.1.2. Cyanogenic glycosides, alkaloids, and saponinsCyanogenic glycosides were present in only 2.3% of species screened. This virtual
absence of cyanogenesis was also recorded in a lowland rain forest in Costa Ricawherein only 25 out of 488 species (5.1%) of woody plants screened were cyanogenic(Thomsen and Brimer, 1997). Cyanogenesis appears to be limited only to certainfamilies such as Leguminosae, Rosaceae, Euphorbiaceae and Passifloraceae (Conn,1979), and cyanogenic glycosides appear to be very much less ubiquitous as defencechemicals than alkaloids, saponins and phenolics. Alkaloids were absent from semi-ripe and ripe fruit, which could reflect the fact that defences that are deterrent topotential seed dispersers need to be minimised (McKey, 1974). Saponins were foundin all plant parts examined. The lowest occurrence of saponins was found in treetwigs. Much more work needs to be done on the distribution of saponins in planttissues, especially given their possible interactions with both condensed andhydrolysable tannins in influencing the potency of these secondary compounds.
4.2. The condensed tannin–hydrolysable tannin interaction
Only in ripe fruit pulp did we find a higher number of species that containedcondensed tannins rather than hydrolysable tannins. The role of hydrolysable tanninsin defence against herbivores has barely been investigated. The seminal and detailedstudies of food selection in primates, especially colobines (e.g. Gartlan et al., 1980;McKey et al., 1981; Waterman et al., 1988; Kool, 1992) have neither quantifiedhydrolysable tannins nor investigated their role in food selection. However, the bark-eating tropical squirrel Sundasciurus lowii was found to select barks with low levelsof hydrolysable tannins (Whitten and Whitten, 1987). Since condensed tannins areprobably not effective deterrents against insect and mammalian herbivores(Waterman and Kool, 1994; Reed, 1995; Ayres et al., 1997) and probably functionlargely as anti-microbial or anti-fungal agents (Waterman, 1983), it is possible thathydrolysable tannins have a more potent action against herbivores than condensedtannins (Swain, 1977; Zucker, 1983; Reed, 1995). Zimmer (1997) found that ingestedgallotannins increased the surface tension of gut fluid, indicating reduced concen-trations of free surfactants, while Barbehenn et al. (1996) found that the gut per-itrophic membrane in polyphagous grasshoppers was easily permeated by severalgallotannins. It is, therefore, interesting that we found that significantly fewer specieshad ripe fruit containing hydrolysable tannins rather than condensed tannins as thesemight deter dispersal agents. However, there are conflicting claims for beneficial andtoxic effects caused by hydrolysable tannins such as ellagitannins in various animalspecies including rodents and ruminants (Clifford and Scalbert, 2000). Similarlyalmost no information is available on the occurrence of gallotannins and ellagitanninsrelative to each other. Our study has shown that gallotannins and ellagitannins arenot significantly segregated in any plant part. Furthermore, of the 31 species thatwere examined for gallotannins and ellagitannins, only 10 species contained both
1234 S. Mali, R.M. Borges / Biochemical Systematics and Ecology 31 (2003) 1221–1246
these compounds, five contained only gallotannins, six only ellagitannins and 10contained neither compound. The biological significance of these findings is as yetunclear and may merely reflect phylogenetic constraints (Gottlieb et al., 1993).
4.3. The alkaloid–tannin interaction
In tropical forests, although alkaloids and phenolics have been widely investigated,few have examined their patterns of co-occurrence (Gartlan et al., 1980; Lebreton,1982; Janzen and Waterman, 1984). Gartlan et al. (1980) found a segregationbetween alkaloids and tannins in mature leaves of Douala-Edea and Kibale forests,while Janzen and Waterman (1984) found a negative correlation between alkaloidand tannin contents in a dry forest in Costa Rica. This is expected as alkaloids andtannins are believed to form insoluble alkaloid-tannates in herbivore guts, thus negat-ing the effects of each other (Freeland and Janzen, 1974). The negative associationbetween alkaloids and tannins was also predicted by Feeny (1976) from apparencytheory. Within plant families, after correcting for species relatedness, Silvertown andDodd (1996) found a negative association between the proportion of species contain-ing tannins and those containing alkaloids. Our results also show that across almostall plant parts, the number of species containing tannins alone was greater than thenumber of species containing both alkaloids and tannins.
4.4. The saponin–tannin interaction
Saponins are widespread in plants and cause haemolysis, enzyme inhibition, andalteration of gut surface tension in herbivores (Applebaum and Kirk, 1979). Althoughthe anti-nutritional effects of saponins in various forages on domesticated herbivores(Klita et al., 1996; Newbold et al., 1997), and the anti-feedant effects of the saponinsof a few plantation tree species on leaf-cutting ants (Folgarait et al., 1996) have beendemonstrated, there has been no investigation of either the community-wide presenceof saponins in tropical forests or of the effects of saponins on tropical forest herbiv-ores. Martin and Martin (1984) showed that detergency negated the anti-digestibilityeffects of tannins in the tobacco hornworm, suggesting that the surfactant propertiesof saponins could function similarly. Freeland et al. (1985) demonstrated that thesimultaneous consumption of tannins and saponins reduced the deleterious effectscaused by the consumption of either saponins or tannins alone. These findings leadto the prediction that saponins and tannins should not co-occur in plant parts, whichis the result that we obtained in this study when we found that within each plantpart category the number of species containing tannins alone was greater than thenumber containing both saponins and tannins. We also found saponins to occur in allplant part categories, as has been found in other studies (Applebaum and Kirk, 1979).
4.5. The alkaloid–saponin interaction
The alkaloid–saponin interaction in herbivore guts and its possible influence onherbivore food selection has scarcely been investigated. Alkaloids and saponins may
1235S. Mali, R.M. Borges / Biochemical Systematics and Ecology 31 (2003) 1221–1246
cause greater deterrence to herbivores when they co-occur than when they occurindependently owing to a synergistic effect, as was found for the seeds ofErythrophleum guineense (Caesalpiniaceae) (Kerharo and Adam, 1974). If this is thegeneral case, then alkaloids and saponins may be expected to co-occur, or at leastthere does not appear to be any biological reason to expect a negative associationbetween these compounds. In our study we were unable to find any clear patternof segregation between alkaloids and saponins and also little evidence of positiveassociation. Much more work is needed in this area.
5. Summary
In summary, we have presented data on the correspondence between protein-preci-pitating assays and chemical tests for tannin activity; we have also measured bothcondensed and hydrolysable tannins (gallotannins and ellagitannins) in a variety ofplant parts, analysed fibre contents and screened for three types of toxins in plantsfrom a wide array of families and orders within a tropical seasonal cloud forestin India.
Acknowledgements
This research was funded by a grant to RMB from the United States Fish andWildlife Service. We thank the Wildlife Institute of India for collaboration. We thankHema Somanathan for helping with data collection and analysis. We are grateful toDoyle McKey for useful suggestions throughout the study, and for helpful commentson this manuscript. We thank Anne Hagerman for providing the quebracho tanninand the protocols for tannin analysis.
Appendix A
1236 S. Mali, R.M. Borges / Biochemical Systematics and Ecology 31 (2003) 1221–1246T
able
A1
Lev
els
ofph
enol
icco
mpo
unds
and
fibre
and
pres
ence
ofal
kalo
ids
and
sapo
nins
intr
ees
and
liana
sat
Bhi
mas
hank
ar
Plan
tpa
rtan
dsp
ecie
sA
stri
ngen
cyT
otal
phen
olic
Con
dens
edG
allo
tann
inE
llagi
tann
inA
DF
ND
FA
DL
Alk
aloi
dsSa
poni
nco
nten
tta
nnin
Mat
ure
leaf
Act
inod
aphn
e–
––
––
––
–A
–an
gust
ifol
iaA
moo
rala
wii
2.4
0.4
00
032
.244
.428
.3A
AA
tala
ntia
race
mos
a–
––
––
––
–P
AB
ride
lia
retu
sa–
––
––
––
–P
AC
anth
ium
dico
ccum
––
––
0–
––
A–
Cas
sine
sp.
00.
20
00
20.3
27.3
15.1
AP
Dio
spyr
osm
onta
na3.
20.
23.
30
0.6
23.0
26.6
22.9
AP
Dio
spyr
ossy
lvat
ica
6.8
0.7
7.6
00
31.0
36.8
20.1
A–
Dip
locl
isia
0.3
1.6
57.2
0.3
0.1
27.6
43.4
7.5
AA
glau
cesc
ensa
Em
beli
ari
besa
1.8
0.6
1.5
00
38.1
42.1
33.8
PA
Fic
usca
llos
a21
.11.
323
.40
041
.543
.832
.2A
AF
icus
race
mos
a–
––
––
––
–A
–F
laco
urti
ain
dica
––
––
––
––
A–
Gar
cini
ata
lbot
ii–
––
––
––
–A
PG
netu
mul
aa2.
01.
81.
20
031
.245
.222
.0A
PL
itse
ast
ocks
ii–
––
––
––
–A
PM
acar
anga
pelt
ata
––
––
0–
––
AA
Mal
lotu
sph
ilip
pens
is6.
61.
14.
70.
10.
238
.957
.630
.1A
AM
angi
fera
indi
ca13
.02.
112
.0–
040
.938
.730
.0A
AM
ayte
nus
roth
iana
0.6
0.2
1.0
00
33.3
43.5
29.1
PA
Mem
ecyl
onum
bell
atum
0.4
––
0.5
026
.232
.711
.1A
AM
ezon
euro
n–
––
––
––
–A
–cu
cull
atum
a
Ole
adi
oica
––
––
––
––
AA
Ran
dia
dum
etor
um–
––
––
––
–P
–R
andi
aru
gulo
saa
13.0
1.1
66.0
0.1
023
.528
.022
.0A
A(c
onti
nued
onne
xtpa
ge)
1237S. Mali, R.M. Borges / Biochemical Systematics and Ecology 31 (2003) 1221–1246T
able
A1
(con
tinu
ed)
Plan
tpa
rtan
dsp
ecie
sA
stri
ngen
cyT
otal
phen
olic
Con
dens
edG
allo
tann
inE
llagi
tann
inA
DF
ND
FA
DL
Alk
aloi
dsSa
poni
nco
nten
tta
nnin
Rou
rea
sant
aloi
desa
––
––
––
––
AA
Sym
ploc
osbe
ddom
ei6.
40.
51.
22.
50
28.2
35.3
17.9
AA
Syzy
gium
cum
ini
––
––
0–
––
AA
Syzy
gium
gard
neri
13.4
1.7
01.
00
29.9
36.5
20.5
AA
Ter
min
alia
bell
eric
a–
––
––
––
–A
PT
erm
inal
iach
ebul
a–
––
––
––
–A
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enti
lago
bom
baie
nsis
a20
.02.
620
.00.
10
32.1
41.6
30.1
A–
Xan
toli
sto
men
tosa
17.6
0.8
14.3
00.
164
.350
.157
.9A
A
Imm
atur
ele
afA
ctin
odap
hne
13.2
0.8
14.0
00
60.6
60.9
58.3
AP
angu
stif
olia
Cas
sine
pani
cula
ta4.
00.
21.
80
050
.539
.330
.6A
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iosp
yros
sylv
atic
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111
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.726
.240
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clis
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959
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033
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sa
Fic
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a18
.62.
656
.70
036
.623
.623
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AG
arci
nia
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17.0
1.5
0.7
00.
337
.032
.135
.5A
PG
netu
mul
aa0.
82.
50.
40
0.2
9.1
28.7
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Lit
sea
stoc
ksii
17.0
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00.
240
.340
.026
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angi
fera
indi
ca0.
53.
07.
01.
30
23.9
28.1
11.6
PA
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ecyl
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atum
0.4
0.8
15.8
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.527
.414
.7A
AO
lea
dioi
ca1.
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03.
20
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.234
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sa0.
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.80.
20.
370
.629
.219
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mpl
ocos
bedd
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0.7
––
3.8
023
.828
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zygi
umcu
min
i22
.511
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31.
335
.134
.818
.0A
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zygi
umga
rdne
ri15
.41.
421
.81.
50.
925
.529
.811
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–T
erm
inal
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ebul
a16
.012
.01.
61.
30.
610
.110
.39.
6A
–V
enti
lago
bom
baie
nsis
a12
.80.
736
.00
022
.432
.016
.6A
A
1238 S. Mali, R.M. Borges / Biochemical Systematics and Ecology 31 (2003) 1221–1246T
able
A1
(con
tinu
ed)
Plan
tpa
rtan
dsp
ecie
sA
stri
ngen
cyT
otal
phen
olic
Con
dens
edG
allo
tann
inE
llagi
tann
inA
DF
ND
FA
DL
Alk
aloi
dsSa
poni
nco
nten
tta
nnin
Petio
leA
ctin
odap
hne
13.2
1.3
11.2
00
42.3
52.0
34.0
A–
angu
stif
olia
Cas
sine
sp.
–0.
11.
00
0–
––
A–
Dio
spyr
ossy
lvat
ica
3.0
0.2
3.6
0.1
0.3
33.4
38.3
19.5
AP
Dip
locl
isia
6.0
0.2
13.2
00
49.1
58.5
43.0
AP
glau
cesc
ensa
Gar
cini
ata
lbot
ii15
.45.
257
.40
029
.732
.912
.9A
PG
netu
mul
aa1.
01.
11.
10.
030
30.6
41.6
30.1
A–
Mac
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gium
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1239S. Mali, R.M. Borges / Biochemical Systematics and Ecology 31 (2003) 1221–1246T
able
A1
(con
tinu
ed)
Plan
tpa
rtan
dsp
ecie
sA
stri
ngen
cyT
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olic
Con
dens
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ula
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ire
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atur
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uit
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ocar
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ire
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ure
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osa
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usre
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ula
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A
1240 S. Mali, R.M. Borges / Biochemical Systematics and Ecology 31 (2003) 1221–1246T
able
A1
(con
tinu
ed)
Plan
tpa
rtan
dsp
ecie
sA
stri
ngen
cyT
otal
phen
olic
Con
dens
edG
allo
tann
inE
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gife
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30
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A–
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gium
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00
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Dio
spyr
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ica
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00
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Dip
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isia
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cini
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20
00
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17.5
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Gne
tum
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Lit
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gife
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ylon
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30
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Ole
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ploc
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ddom
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5A
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4A
PSy
zygi
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rdne
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25.
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min
alia
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ula
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––
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eria
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osa
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00
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atur
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sa
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A
1241S. Mali, R.M. Borges / Biochemical Systematics and Ecology 31 (2003) 1221–1246T
able
A1
(con
tinu
ed)
Plan
tpa
rtan
dsp
ecie
sA
stri
ngen
cyT
otal
phen
olic
Con
dens
edG
allo
tann
inE
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tann
inA
DF
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FA
DL
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Ter
min
alia
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ula
11.3
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0.8
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i-m
atur
ese
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nia
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0.5
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310
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1242 S. Mali, R.M. Borges / Biochemical Systematics and Ecology 31 (2003) 1221–1246T
able
A1
(con
tinu
ed)
Plan
tpa
rtan
dsp
ecie
sA
stri
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cyT
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olic
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dens
edG
allo
tann
inE
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tann
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1243S. Mali, R.M. Borges / Biochemical Systematics and Ecology 31 (2003) 1221–1246T
able
A1
(con
tinu
ed)
Plan
tpa
rtan
dsp
ecie
sA
stri
ngen
cyT
otal
phen
olic
Con
dens
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allo
tann
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00
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00
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10
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toli
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men
tosa
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ire
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–
For
para
met
ers
othe
rth
anal
kalo
ids
and
sapo
nins
,va
lues
are
expr
esse
das
follo
ws:
astr
inge
ncy
and
tota
lph
enol
icco
nten
tsas
perc
ent
tann
icac
id,
cond
ense
dta
nnin
sas
perc
ent
queb
rach
ota
nnin
,ga
llota
nnin
sas
perc
ent
galli
cac
id,
and
ella
gita
nnin
sas
perc
ent
ella
gic
acid
ondr
yw
eigh
tba
sis.
Onl
yal
kalo
ids
and
sapo
nins
wer
equ
alita
tivel
yde
tect
ed,
whe
rein
‘P’
indi
cate
spr
esen
ce;
‘A’
indi
cate
sab
senc
ean
d–
indi
cate
sva
lues
not
estim
ated
;A
DF,
acid
dete
rgen
tfib
re;
ND
F,ne
utra
lde
terg
ent
fibre
;A
DL
,ac
idde
terg
ent
ligni
n.C
yano
geni
cgl
ycos
ides
wer
eab
sent
inal
lth
ese
item
san
dhe
nce
not
show
n.a
Indi
cate
slia
na.
1244 S. Mali, R.M. Borges / Biochemical Systematics and Ecology 31 (2003) 1221–1246
References
Applebaum, S.W., Kirk, Y., 1979. Saponins. In: Rosenthal, G.A., Janzen, D.H. (Eds.), Herbivores. TheirInteraction with Secondary Plant Metabolites. Academic Press, New York, pp. 539–566.
Asquith, T.N., Butler, L.G., 1985. Use of dye-labeled protein as spectrophotometric assay for proteinprecipitants such as tannin. J. Chem. Ecol. 11, 1535–1544.
Ayres, M.P., Clausen, T.P., MacLean, S.E. Jr., Redman, A.M., Reichardt, P.B., 1997. Diversity of structureand antiherbivore activity in condensed tannins. Ecology 78, 1696–1712.
Azaizeh, H.A., Petit, R.E., Sarr, B.A., Phillips, T.D., 1990. Effect of peanut tannin extracts on growth ofAspergillus parasiticus and aflatoxin production. Mycopathologia 110, 125–132.
Barbehenn, R.V., Martin, M.M., Hagerman, A.E., 1996. Reassessment of the roles of the peritrophicenvelope and hydrolysis in protecting polyphagous grasshoppers from hydrolysable tannins. J. Chem.Ecol. 22, 1901–1919.
Berenbaum, M.R., 1995. The chemistry of defence: theory and practice. Proc. Natl. Acad. Sci. USA 92,2–8.
Borges, R.M., 1989. Resource heterogeneity and the foraging ecology of the Malabar Giant SquirrelRatufa indica. PhD dissertation. University of Miami, Florida.
Borges, R.M., 1990. Sexual and site differences in calcium consumption by the Malabar giant squirrelRatufa indica. Oecologia 85, 80–86.
Borges, R.M., 1992. A nutritional analysis of foraging in the Malabar giant squirrel (Ratufa indica). Biol.J. Linn. Soc. 47, 1–21.
Borges, R.M., 1993. Figs and Malabar giant squirrels in two tropical forests in India. Biotropica 25,183–190.
Clifford, M.N., Scalbert, A., 2000. Ellagitannins. Nature, occurrence and dietary burden. J. Sci. FoodAgric. 80, 1118–1125.
Conn, E.E., 1979. Cyanide and cyanogenic glycosides. In: Rosenthal, G.A., Janzen, D.H. (Eds.), Herbiv-ores. Their Interaction with Secondary Plant Metabolites. Academic Press, New York, pp. 387–412.
Davies, A.G., Bennett, E.L., Waterman, P.G., 1988. Food selection by the South-east Asian colobinemonkeys (Presbytis rubicunda and Presbytis melalophos) in relation to plant chemistry. Biol. J. Linn.Soc. 34, 33–56.
Feeny, P., 1976. Plant apparency and chemical defense. In: Wallace, J.W., Mansell, R.L. (Eds.), Biochemi-cal Interactions between Plants and Insects. Recent Advances in Phytochemistry, vol. 10. PlenumPress, New York, pp. 1–40.
Folgarait, P.J., Dyer, L.A., Marquis, R.J., Braker, H.E., 1996. Leaf-cutting ant preferences for five tropicalplantation tree species growing under different light conditions. Entomol. Exp. Appl. 80, 521–530.
Freeland, W.J., Janzen, D.H., 1974. Strategies of herbivory in mammals: the role of plant secondarycompounds. Am. Nat. 108, 269–289.
Freeland, W.J., Calcott, P.H., Anderson, L.R., 1985. Tannins and saponin: interaction in herbivore diets.Biochem. Syst. Ecol. 13, 189–193.
Gartlan, J.S., McKey, D.B., Waterman, P.G., Mbi, C.N., Struhsaker, T.T., 1980. A comparative study ofthe phytochemistry of two African rainforests. Biochem. Syst. Ecol. 8, 401–422.
Goering, H.K., van Soest, P.J., 1970. Forage fiber analyses (apparatus, reagents, procedures, and someapplications). USDA Agricultural Handbook No. 379. Agricultural Research Service.
Goldstein, J.L., Swain, T., 1963. Changes in tannins in ripening fruits. Phytochemistry 2, 371–383.Gottlieb, O.R., Kaplan, M.A.C., Zocher, D.H.T., 1993. A chemosystematic overview of Magnoliidae,
Ranunculidae, Caryophyllidae and Hamamelidae. In: Kubitzki, K., Rohwer, J.G., Bittrich, V. (Eds.),The Families and Genera of Vascular Plants, vol. II. Flowering Plants. Dicotelydons. Magnoliid, Ham-amelid and Caryophyliid Families. Springer, Berlin, pp. 20–33.
Gottlieb, O.R., Borin, M.R.M.B., Kaplan, M.A.C., 1995. Biosynthetic interdependence of lignins andsecondary metabolites in angiosperms. Phytochemistry 40, 99–113.
Hagerman, A.E., Butler, L.G., 1978. Protein precipitation method for the quantitative determination oftannins. J. Agric. Food Chem. 26, 809–812.
Haslam, E., 1985. Metabolites and Metabolism: A Commentary on Secondary Metabolism. Oxford Uni-versity Press, Oxford.
1245S. Mali, R.M. Borges / Biochemical Systematics and Ecology 31 (2003) 1221–1246
Inoue, K.H., Hagerman, A.E., 1988. Determination of gallotannins with rhodanine. Anal. Biochem. 169,363–369.
Janzen, D.H., Waterman, P.G., 1984. A seasonal census of phenolics, fibre and alkaloids in foliage offorest trees in Costa Rica: some factors influencing their distribution and relation to host selection bySphingidae and Saturniidae. Biol. J. Linn. Soc. 21, 439–454.
Kerharo, J., Adam, J.G., 1974. La Pharmacopee Senegalaise Traditionelle. Vigot, Paris.Klita, P.T., Mathison, G.W., Fenton, T.W., Hardin, R.T., 1996. Effect of alfalfa root saponins on digestive
function in sheep. J. Anim. Sci. 74, 1144–1156.Kool, K.M., 1992. Food selection by the silver leaf monkey, Trachypithecus auratus sondaicus, in relation
to plant chemistry. Oecologia 90, 527–533.Kubitzki, K., Gottlieb, O.R., 1984. Phytochemical aspects of angiosperm origin and evolution. Acta Bot.
Neerl. 33, 457–468.Lebreton, P., 1982. Tannins ou alcaloides: deux tactiques phytochimiques de disuasion des herbivores.
Rev. Ecol. (Terre Vie) 36, 539–572.Mali, S., 1998. Plant chemical profiles and their influence on food selection in the Malabar giant squirrel
Ratufa indica. PhD dissertation. University of Bombay, India.Martin, M.M., Martin, J.S., 1982. Tannin assays in ecological studies: lack of correlation between phe-
nolics, proanthocyanidins, and protein precipitation constituents of mature foliage in six oak species.Oecologia 54, 205–211.
Martin, M.M., Martin, J.S., 1984. Surfactants: their role in preventing the precipitation of proteins bytannins in insect guts. Oecologia 61, 342–345.
McKey, D., 1974. Adaptive patterns in alkaloid physiology. Am. Nat. 108, 305–320.McKey, D.B., Waterman, P.G., Mbi, C.N., Gartlan, J.S., Struhsaker, T.T., 1978. Phenolic content of
vegetation in two African rain forests: ecological implications. Science 202, 61–64.McKey, D.B., Gartlan, J.S., Waterman, P.G., Choo, G.M., 1981. Food selection by black colobus monkeys
(Colobus satanas) in relation to food chemistry. Biol. J. Linn. Soc. 16, 115–146.Milton, K., 1979. Factors influencing leaf choice by howler monkeys: a test of some hypotheses of food
selection by generalist herbivores. Am. Nat. 114, 362–378.Mole, S., Waterman, P.G., 1987a. A critical analysis of techniques for measuring tannins in ecological
studies. I. Techniques for chemically defining tannins. Oecologia 72, 137–147.Mole, S., Waterman, P.G., 1987b. A critical analysis of techniques for measuring tannins in ecological
studies. II. Techniques for biochemically defining tannins. Oecologia 72, 148–156.Mole, S., Butler, L.G., Hagerman, A.E., Waterman, P.G., 1989. Ecological tannin assays: a critique.
Oecologia 78, 93–96.Newbold, C.J., El-Hassan, S.M., Wang, J., Ortega, M.E., Wallace, R.J., 1997. Influence of foliage from
African multipurpose trees on activity of rumen protozoa and bacteria. Br. J. Nutr. 78, 237–249.Oates, J.F., Waterman, P.G., Choo, G.M., 1980. Food selection by the south Indian leaf-monkey Presbytis
johnii, in relation to leaf chemistry. Oecologia 45, 45–56.Ossipov, V., Loponen, J., Ossipova, S., Haukioja, E., Pihlaja, K., 1997. Gallotannins of birch Betula
pubescens leaves: HPLC separation and quantification. Biochem. Syst. Ecol. 25, 493–504.Porter, L.J., Hrstich, L.N., Chan, B.C., 1986. The conversion of procyanidins and prodelphinidins to
cyanidin and delphinidin. Phytochemistry 25, 223–230.Reed, J.D., 1995. Nutritional toxicology of tannins and related polyphenols in forage legumes. J. Anim.
Sci. 73, 1516–1528.Rhoades, D.F., Cates, R.G., 1976. Toward a general theory of plant antiherbivore chemistry. Recent Adv.
Phytochem. 10, 168–213.Saldanha, C.J., 1984. Flora of Karnataka, vol. I. Oxford & IBH, New Delhi.Saldanha, C.J., 1986. Flora of Karnataka, vol. II. Oxford & IBH, New Delhi.Saldanha, C.J., Nicolson, D.H., 1976. Flora of Hassan District. Karnataka, India. Amerind Publishing
Co., New Delhi.Sawa, T., Mayami, N., Akaike, T., Ono, K., Maeda, H., 1999. Alkylperoxyl radical-scavenging activity
of various flavonoids and other phenolic compounds: implications for the anti-tumor-promoter effectof vegetables. J. Agric. Food Chem. 47, 397–402.
1246 S. Mali, R.M. Borges / Biochemical Systematics and Ecology 31 (2003) 1221–1246
Silvertown, J., Dodd, M., 1996. Comparing plants and connecting traits. Philos. Trans. R. Soc. Lond. BBiol. Sci. 351, 1233–1239.
Singleton, V.L., Rossi, J.A., 1965. Colorimetry of total phenolics with phosphomolybdic phototungsticacid reagents. Am. J. Enol. Vitic. 16, 144–158.
Sokal, R.R., Rohlf, F.J., 1981. Biometry. W.H. Freeman, San Francisco.Swain, T., 1977. Secondary compounds as protective agents. Annu. Rev. Plant Physiol. 28, 479–501.Thomsen, K., Brimer, L., 1997. Cyanogenic constituents in woody plants in natural lowland rain forest
in Costa Rica. Bot. J. Linn. Soc. 124, 273–294.Trease, G.E., Evans, W.C., 1972. Pharmacognosy. Balliere Tindall, London.Waterman, P.G., 1983. Distribution of secondary metabolites in rain forest plants: towards an understand-
ing of cause and effect. In: Sutton, S.L., Whitmore, T.C., Chadwick, A.C. (Eds.), Tropical Rain Forest:Ecology and Management. Blackwell, Oxford, pp. 167–179.
Waterman, P.G., 1984. Food acquisition and processing as a function of plant chemistry. In: Chivers,D.J., Wood, B.A., Bilsborough, A. (Eds.), Food Acquisition and Processing in Primates. Plenum Press,New York, pp. 177–211.
Waterman, P.G., Choo, C.M., 1981. The effects of digestibility-reducing compounds in leaves on foodselection by some Colobinae. Malays. Appl. Biol. 10, 147–162.
Waterman, P.G., Kool, K.M., 1994. Colobine food selection and food chemistry. In: Davies, A.G., Oates,J.F. (Eds.), Colobine Monkeys. Their Ecology, Behaviour and Evolution. Cambridge University Press,Cambridge, pp. 251–284.
Waterman, P.G., Mole, S., 1989. Extrinsic factors influencing production of secondary metabolites inplants. In: Bernays, E.A. (Ed.), Insect–Plant Interactions. CRC Press, Boca Raton, FL, pp. 107–134.
Waterman, P.G., Mole, S., 1994. Analysis of Phenolic Plant Metabolites. Blackwell, Oxford.Waterman, P.G., Ross, J.A.M., Bennett, E.L., Glyn Davies, A., 1988. A comparison of the floristics and
leaf chemistry of the tree flora in two Malaysian rain forests and the influence of leaf chemistry onpopulations of colobine monkeys in the Old World. Biol. J. Linn. Soc. 34, 1–32.
Whitten, J.E.J., Whitten, A.J., 1987. Analysis of bark-eating in a tropical squirrel. Biotropica 19, 107–115.Wilson, T.C., Hagerman, A.E., 1990. Quantitative determination of ellagic acid. J. Agric. Food Chem.
38, 1678–1683.Zimmer, M., 1997. Surfactants in the gut fluids of Porcellio scaber (Isopoda: Oniscidea), and their interac-
tions with phenolics. J. Insect Physiol. 43, 1009–1014.Zucker, W.V., 1983. Tannins: does structure determine function? Am. Nat. 121, 335–365.