Biochemical Systematics and Ecology 31 (2003) 1221–1246 www.elsevier.com/locate/biochemsyseco Phenolics, fibre, alkaloids, saponins, and cyanogenic glycosides in a seasonal cloud forest in India S. Mali a, R.M. Borges b,∗ a National Innovation Foundation, Vastrapur, Ahmedabad 380 015, India b Centre for Ecological Sciences, Indian Institute of Science, Bangalore 560 012, India Received 18 June 2001; accepted 3 February 2003 Abstract We 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 screened for alkaloids, saponins and cyanogenic glycosides in 271 plant parts across 33 tree and 10 liana 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 of Bridelia retusa (Euphorbiaceae), i.e. in 2.3% of species examined. Alkaloids were absent from petioles, ripe fruit and mature seeds examined. Saponins were found in all types of plant parts. Condensed tannins occurred in almost all plant parts examined (93.6%), while hydrolysable tannins were less ubiquitous (gallotannins in 31.2% of samples, and ellagitannins in 18.9%). Astringency levels were significantly correlated with total phenolic, condensed tannin, and hydrolysable tannin contents. Condensed tannin and hydrolysable tannin contents were not related. Immature leaves, 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 each plant part category, the frequency of species containing tannins together with alkaloids or saponins was significantly lower than the frequency of species containing tannins alone. There was, however, no segregation between alkaloids and saponins. 2003 Published by Elsevier Science Ltd. Keywords: Condensed tannins; Hydrolysable tannins; Plant defences; Plant secondary compounds; Ratufa indica; 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 phenolics and alkaloids) are available for only a few tropical forests in Africa and Asia (McKey et al., 1978; Gartlan et al., 1980; McKey et al., 1981; Davies et al., 1988; Waterman et al., 1988; Kool, 1992). Especially with regard to phenolics, most of these studies have focused on only a few estimation methods. For example, most studies have not examined hydrolysable tannins or the relationship between condensed and hydrolys- able tannins in plant parts. In this paper, we report on a quantitative analysis of phenolics, condensed tannins, hydrolysable tannins (gallotannins and ellagitannins), bovine serum albumin (BSA) assays for tannin astringency, digestibility reducers such as acid detergent fibre (ADF), neutral detergent fibre (NDF) and acid detergent lignin (ADL) for 271 ephemeral and non-ephemeral parts of 33 tree and 10 liana species within a seasonal cloud forest community in the Western Ghats of India. We also provide qualitative data on saponins, alkaloids, and cyanogenic glycosides for these resources. The sampled tree and liana species constituted more than 90% of the species at the site, and the samples were collected as part of a larger study on the foraging strategy of the Malabar giant squirrel Ratufa indica. We restricted our analysis to those compounds that have been found to affect the foraging strategy of arboreal mammalian herbivores such as primates (e.g. Oates et al., 1980; McKey et al., 1981; Waterman and Choo, 1981; Waterman and Kool, 1994) and giant squirrels (Borges, 1989; Borges, 1992). Data on the macro- and micro-nutrients within these plant parts will be presented elsewhere. Because of the enormous structural diversity and lack of general techniques, we restricted our analysis to the qualitative analysis of toxins. Owing to the structural diversity of tannins and the procedural difficulties involved in their quantitative analysis (Martin and Martin, 1982; Mole and Water- man, 1987a,b; Mole et al., 1989; Waterman and Mole, 1989), we employed only standard and improved methods recommended by Waterman and Mole (1994).We also used a combination of chemical and protein-precipitating methods to determine the biological activity of tannins. Therefore, our results are comparable with studies of other forest communities done elsewhere. Despite the limitations of our data set, in terms of missing analyses for some metabolites 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 attempt in this paper to examine the co-occurrence of metabolites such as tannins, alkaloids and saponins in various plant parts in order to examine predictions about the possible synergisms or negative interactions between these metabolites. For example, since alkaloids and tannins react to form insoluble alkaloid-tannates in herbivore guts pre- venting reactions between tannins and proteins (Freeland and Janzen, 1974), and because the surfactant properties of saponins negate the anti-digestibility effects of tannins (Martin and Martin, 1984; Freeland et al., 1985), tannins are expected not to 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 be expected to co-occur to enhance herbivore deterrence. In this paper we therefore primarily report on the distribution of secondary com- S. Mali, R.M. Borges / Biochemical Systematics and Ecology 31 (2003) 1221–1246 1223 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 Maharashtra State, India (19°21Ј–19°11ЈN, 73°31Ј–73°37ЈE, altitude 900 m, annual precipitation 3000 mm). This is a species-poor forest where only eight tree species contribute to 85.4% of relative dominance values, and the three most common species (Mangifera indica [Anacardiaceae], Memecylon umbellatum [Melastomataceae] and Olea dioica [Oleaceae]) contribute to 64.1% of relative dominance (Table 1). The forest is highly fragmented; our study site was situated in the largest and best protected fragment constituting 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 thus covered a wide spectrum of species within the cloud forest. All analysed species with only two exceptions (Terminalia bellerica and Terminalia chebula) were part of the natural evergreen community of the cloud forest. As these chemical analyses were performed as part of a larger study on the foraging ecology of the giant squirrel R. indica (Mali, 1998), our sampling and choice of chemical analyses were designed to understand food preference and food avoidance, and were also influenced by the availability of adequate sample and other logistic constraints. Samples of both ephemeral and non-ephemeral items were collected at the time of year when they featured most in the diet of the giant squirrel (to control for seasonal variation in phytochemistry, 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 and cyanogenic glycosides We field-tested for alkaloids using Dragendorff’s and Mayer’s reagents, and later performed confirmatory tests on dried material (Gartlan et al., 1980). For saponin detection, we vigorously agitated small aqueous extracts with distilled water and took 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 picrate test to detect cyanogenic glycosides (Conn, 1979). 2.4. Quantitative phytochemical analysis The results of the chemical analyses presented here are single values estimated from 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 1 Orders, families and species of trees and lianas for which phytochemical data were obtained and relative dominance of trees in the study site Order Family Species Relative dominance Trees Celastrales 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 0.04 Lamiales Oleaceae Olea dioica Roxb.b 14.3 Laurales Lauraceae Actinodaphne angustifolia