phenolics, ﬁbre, alkaloids, saponins, and cyanogenic glycosides...

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).


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)


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,


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).


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.


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

).


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


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


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


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

).


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


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


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

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and

pres

ence

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intr

ees

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liana

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ure

leaf

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01.

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1237S. Mali, R.M. Borges / Biochemical Systematics and Ecology 31 (2003) 1221–1246T

able

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(con

tinu

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