Ecology, 78(3), 1997, pp. 782–798
q 1997 by the Ecological Society of America
WHY ARE SOME FRUITS TOXIC? GLYCOALKALOIDS IN SOLANUM
AND FRUIT CHOICE BY VERTEBRATES
MARTIN L. CIPOLLINI1
AND
DOUGLAS J. LEVEY
Department of Zoology, University of Florida, 223 Bartram Hall, Gainesville, Florida 32611 USA
Abstract. We examined the influence of secondary metabolites (glycoalkaloids) of
Solanum fruits on fruit consumption by seed dispersers and seed predators. Our goal was
to determine the degree to which secondary metabolites might explain fruit–frugivore interactions that heretofore have not been explained by other fruit-related variables (e.g.,
color, nutrient content, seediness, etc.). Using feeding trails with real fruits and artificial
fruit pulp media, we tested two sets of hypotheses, one relating to the specificity of secondary metabolites and the other to intra- and interspecific variation in secondary metabolite
content. First, the Directed Toxicity Hypothesis was tested against its alternative, the General
Toxicity Hypothesis. The Directed Toxicity Hypothesis posits that fruit pulp secondary
metabolites are directed primarily toward organisms that destroy seeds following fruit
consumption, remaining nontoxic to organisms that disperse seeds. The General Toxicity
Hypothesis states that these secondary metabolites are general in their effects. Second, the
Removal Rate Hypothesis was tested against its alternative, the Nutrient–Toxin Titration
Hypothesis. The Removal Rate Hypothesis posits that profitable fruits, with high natural
rates of removal, contain only low levels of secondary metabolites as defenses against seed
predators and pathogens. In contrast, the Nutrient–Toxin Titration Hypothesis states that
profitable fruits are better able to afford protection by high levels of secondary metabolites
because high nutrient levels compensate for deterrent effects of secondary metabolites.
We compared Solanum carolinense, with high levels of secondary metabolites (glycoalkaloids) in ripe fruit, and S. americanum, with negligible levels, conducting four sets
of feeding trials: (1) Real Fruit trials to determine the preferences of our study animals for
real fruit of each plant species; (2) Fruit Mimic trials using artificial fruit pulp media to
determine the relative effects of nutrient and glycoalkaloid content on fruit preferences;
(3) Interaction trials using artificial fruit pulp media to examine relative and interactive
effects of the two main glycoalkaloids in our fruits ( a-solasonine and a-solamargine); and
(4) Nutrient–Toxin Titration trials using artificial fruit pulp media to determine how much
nutrient content might compensate for deterrent effects.
Both seed predators and seed dispersers preferred the low-glycoalkaloid S. americanum
fruits and were deterred by glycoalkaloid levels typical of ripe S. carolinense fruits. Because
of the generality of the deterrent effects, we rejected the Directed Toxicity Hypothesis.
Additionally, we found no evidence for differences between the two glycoalkaloids in their
deterrent effects, and no evidence of synergism between them. Nutrient effects were generally small; we found no evidence that nutrient variation could compensate for the deterrence produced by levels of glycoalkaloids found in ripe S. carolinense fruits. Ripe fruits
of the presumably more profitable, preferred S. americanum contain much lower levels of
glycoalkaloid. Taken together, our results support the Removal Rate Hypothesis. Secondary
metabolites of ripe fleshy fruits are an important determinant of fruit use by frugivores.
We suggest that a primary function of these metabolites is defense against pests and pathogens. Thus, these organisms should be considered an important influence on fruit–seed
disperser interactions.
Key words: antifungal compounds; antiherbivore compounds; chemical defenses; fruit–seed disperser interactions; fruit pulp nutrients; secondary plant metabolites; seed dispersal; Solanum americanum; Solanum carolinense.
INTRODUCTION
A central issue in studies of fruit–seed disperser interactions is the degree to which reciprocal selection
Manuscript received 23 August 1995; revised 16 May
1996; accepted 11 July 1996; final version received 2 August
1996.
1 Present address: Biology Department, 430 Berry College,
Mount Berry, Georgia 30149 USA.
pressures between fruiting plants and frugivores have
influenced morphological, chemical, and behavioral
traits in each group (Herrera 1985, 1986, Jordano 1987,
Howe 1992, Stiles and Rosselli 1992). A basic assumption is that predictable differences in quality of
seed dispersal among groups of frugivores can explain
variation in fruit traits (Schupp 1992). Yet, most attempts to delineate such groups have been only mar-
782
April 1997
EFFECTS OF GLYCOALKALOIDS IN SOLANUM FRUITS
ginally successful or have not fully incorporated phylogeny (e.g., Gautier-Hion et al. 1985, Debussche et al.
1987, Debussche and Isenmann 1989, Willson et al.
1989, Gautier-Hion 1990). In fact, recent studies using
rigorous phylogenetic null models have failed to support the notion that frugivores influence variation in
fruit traits (Herrera 1987, 1992, Jordano 1987, Bremer
and Eriksson 1992). Although some data suggest congruence between the type of fruit ingested and the digestive physiology of particular frugivores (e.g., Martinez del Rio et al. 1989), these studies cannot be taken
as proof that frugivores have influenced fruit traits. Not
only might the converse be true (frugivores may adapt
to independently evolving fruit traits), but ecological
‘‘fitting’’ processes might also explain such patterns,
since animal species might utilize only those fruits to
which they have been pre-adapted (cf., Janzen 1985).
We suggest that most previous studies of fruit–seed
disperser interactions have been limited by focusing
solely upon vertebrate seed dispersers. Many potentially detrimental organisms may compete with legitimate seed dispersers for fruit resources (Janzen 1977,
Herrera 1982). Their effects on the evolution of fruit
traits will be different than, and even opposite to, the
effects of seed dispersers. Such organisms include vertebrate seed-predators, insect fruit- and seed-predators,
and microbial fruit-rot agents. In contrast with organisms that defecate, or otherwise disperse, a majority of
ingested seeds in viable condition, such organisms kill
nearly all of the seeds in the process of fruit consumption and digestion. Viewed in this context, plants
are faced with an evolutionary dilemma: how to make
fruits simultaneously attractive to seed-dispersing frugivores and unattractive to all other frugivores. Fruit
secondary metabolites are probably involved as mediators of this conflict (Janzen 1977, Herrera 1982,
Sorensen 1983, Cipollini and Stiles 1992).
Understanding the nature of fruit secondary metabolites and their effects upon various fruit consumers
will help to resolve an apparent evolutionary paradox:
ripe fruits that are apparently poisonous to potential
dispersers (e.g., Atropa belladonna L. and some Solanum spp.). In the same way that recognition of defensive secondary metabolites revolutionized studies
of plant–herbivore and plant–pathogen interactions
(Rhoades and Cates 1976, Rhoades 1979, 1985), we
hope that study of such compounds will yield a new
perspective on fruit–seed disperser interactions. Aside
from several theoretical papers (Herrera 1982, Izhaki
and Safriel 1989, 1990, Mack 1990, Sedinger 1990),
this area has been practically ignored. In fact, with few
exceptions, secondary metabolite composition of ripe
fruit remains unexplored (Goldstein and Swain 1963,
Dement and Mooney 1974, Janzen 1975, 1979, 1983,
Wrangham and Waterman 1983, Ballington et al. 1988,
Gargiullo and Stiles 1991, Cipollini and Stiles 1993).
Here, we develop and test alternative hypotheses
about the role of glycoalkaloids in Solanum fruits. Gly-
783
coalkaloids, which are virtually restricted to the Solanaceae, are composed of C27 steroid aglycones to
which various branched carbohydrate moieties are attached (Ripperger and Schreiber 1981). The best known
compounds are the potato glycoalkaloids, a-solanine
and a-chaconine (Gregory 1984, Van Gelder 1990), but
the structurally related compounds a-solasonine and asolamargine occur more broadly among Solanum species (Ripperger and Schreiber 1981). Glycoalkaloids
can be highly toxic to vertebrates; in some species,
their concentration in ripe fruit (up to 7% of dry mass)
should be high enough to cause lethal effects in a 1–
2 kg vertebrate after consumption of ,10 fruits (based
on an estimate of a 100–1000 mg/kg lethal dose in
clinical and case studies with domestic animals and
humans; Zitnak 1979, Van Gelder 1990). To our knowledge, neither toxicity tests nor feeding-preference trials
have been conducted on wild animals using these or
any other glycoalkaloids.
To test our hypotheses, we used feeding trials on two
species of birds (American Robin, Turdus migratorius;
Northern Bobwhite, Tetrao virginianus) and two species of mammals (Virginia opossum, Didelphis marsupialis; deer mouse, Peromyscus maniculatus). These
species were selected because they are common consumers of Solanum fruits (Martin et al. 1951; M. Cipollini, personal observations) and because they presumably differ in their treatment of seeds. American
Robins and opossums are likely to be seed dispersers,
whereas Northern Bobwhites and deer mice are likely
to be seed predators. We first conducted seed viability
tests on defecated seeds to confirm these presumed differences in seed treatment. We then conducted one set
of feeding trials with real fruit to determine natural
preferences, and three sets of feeding trials with artificial fruits to tease apart the influence of nutrient and
glycoalkaloid content on preference patterns.
Hypotheses
We present two sets of hypotheses. The first set addresses the specificity of fruit pulp secondary metabolites, which may depend on how predictably frugivores differ in their treatment of seeds. The second set
addresses whether or not removal rates of fruits and
pulp nutrient content can explain patterns of intra- and
interspecific variation in secondary metabolites of fruit
pulp.
Secondary metabolite specificity: alternative
hypotheses
1. Directed Toxicity Hypothesis.—Predictable variation among fruit consumers in their effects upon seed
dispersal has resulted in selection for specificity in the
toxicity of secondary metabolites of ripe fruit. Thus,
secondary metabolites of unripe fruit should be directed toward all consumers, whereas those of ripe fruit
should be directed only toward nondispersing consumers (seed predators). A form of this hypothesis was
784
MARTIN L. CIPOLLINI AND DOUGLAS J. LEVEY
proposed initially by Janzen (1975, 1979), who suggested that secondary metabolites within fruit pulp may
‘‘filter out’’ or repel only groups of consumers that
negatively affect seed dispersal. This hypothesis is distinct from the ‘‘optimal defense theory’’ (Rhoades and
Cates 1976), in that no relationship is presumed between ‘‘generalist’’ and ‘‘specialist’’ herbivores and
type of secondary metabolite (e.g., ‘‘quantitative’’ vs.
‘‘qualitative’’ chemicals). Fruit–frugivore interactions
differ fundamentally from plant–herbivore interactions
because of the positive effects of fruit consumption by
particular animals (seed dispersers), which can contrast
markedly with the negative effects of other organisms
(seed predators).
2. General Toxicity Hypothesis.—Unpredictable or
indistinguishable variation among fruit consumers in
their effects upon seed dispersal has resulted in general
toxicity of secondary metabolites within ripe fruit.
Thus, seed-dispersing and non-seed-dispersing frugivores will be affected equally and adversely by pulp
secondary compounds in both ripe and unripe fruit.
This hypothesis suggests that retention of toxins in ripe
fruits represents a trade-off between defense against
damaging organisms and attraction of dispersers (Herrera 1982). In short, ‘‘diffuse’’ interactions between
frugivores and fruiting plants constrain selection for
directed toxicity.
Although little is known about the biological activities of the compounds found in our study fruits (asolasonine and a-solamargine), particularly in a natural
context, we note that toxic effects of the structurally
similar potato glycoalkaloids solanine and chaconine
may differ, and that toxicity thresholds may vary
among vertebrate (Zitnak 1979, Baker et al. 1989), insect (Gregory 1984, Tingey 1984), and fungal species
(McKee 1959, Nes et al. 1983, Rowan et al. 1983,
Gregory 1984, Roddick 1986, 1987, Van Gelder 1990).
Although glycoalkaloids vary in their toxicity among
groups of animals (thus lending credence to the Directed Toxicity Hypothesis), their initial mode of activity is somewhat general. Initial toxic symptoms are
assumed to result from sterol binding (cholesterol, ergosterol, phytosterol) and loss of lipid membrane integrity (Alauddin and Martin-Smith 1962, Zitnak 1979,
Nes et al. 1983, Gregory 1984, Tingey 1984, Sidhu and
Oakenfull 1986, Roddick 1987). For vertebrates, arthropods, and fungi alike, sterol binding results in altered membrane permeability and tissue damage (Zitnak 1979, Roddick 1986). However, variation among
organisms in their sensitivity to glycoalkaloids may
depend upon the binding affinity of the glycoalkaloid
for particular sterols, as well as the quality and quantity
of sterol within the cell membrane (Roddick 1987, Roddick et al. 1990, Keukens et al. 1992). Secondary toxic
symptoms presumably result from interference with
acetylcholinesterase function, which can produce fatal
neurological effects in vertebrates (Zitnak 1979, Roddick 1989). Because nearly all studies of glycoalkaloid
Ecology, Vol. 78, No. 3
toxicity have used intravenous or intraperitoneal injection (Van Gelder 1990), the mechanisms by which
vertebrates might vary in their overall sensitivity to
naturally occurring dietary glycoalkaloids are unclear.
Intra- and interspecific variation in ripe fruit
chemistry: alternate hypotheses
1. Removal Rate Hypothesis.—The degree to which
a particular plant defends its fruits against pests and
pathogens via secondary metabolites depends primarily
on the removal rate of ripe fruit. Fruits that are removed
quickly and predictably by frugivores should be under
little selection pressure for persistence. Conversely,
fruits that are removed slowly and unpredictably should
be under greater selection pressure for defense. Thus,
we predict that plants bearing fruits of high nutritional
quality or fruits that are, for other reasons, removed
quickly and predictably, will show lower levels of secondary metabolites than those plants bearing fruits that
are removed slowly.
2. Nutrient–Toxin Titration Hypothesis.—Due to palatability reduction that results from secondary metabolites, the level of secondary metabolites within ripe
fruit should be positively related to nutritional quality.
As a means of maintaining acceptability, low-nutrient
fruits should be under selection to retain low levels of
unpalatable secondary metabolites. Conversely, fruits
with high nutritional quality should be profitable
enough to allow the retention of higher levels of defenses. Thus, we predict that plants bearing fruits with
high nutritional quality will show higher levels of secondary metabolites than plants with fruits of low nutritional quality. Additionally, feeding trials with frugivores will show that high nutritional quality can compensate for high levels of toxins.
MATERIALS
AND
METHODS
Plant species
An advantage of working with Solanum is that secondary metabolites in this genus are well known, due
to the agricultural importance of several species (e.g.,
potato, S. tuberosum L., and tomato, S. lycopersicum
L.). Of particular interest is the propensity for immature
Solanum fruits to accumulate high levels of alkaloids
(1–7% dry mass); levels that may or may not drop
during fruit maturation. In particular, although most
ripe Solanum fruits contain little or no glycoalkaloid
(Briggs et al. 1961, Schrieber 1963, Carle 1981), fruits
of several species retain levels nearly equivalent to, or
even exceeding, that of unripe fruits (e.g., S. melongena
L., Aubert et al. 1989a, b; S. tuberosum L. and S. xanthocarpum, Zitnak 1979; S. viarum and S. lacinatum,
Miller and Davies 1979; S. aculeatissimum (khasianum) Jacq., Mahato et al. 1980, Nes et al. 1980; S. pseudocapsicum L., Mangla and Kamal 1989).
We focus on two contrasting Solanum species that
are abundant in central Florida, S. americanum Mill,
April 1997
EFFECTS OF GLYCOALKALOIDS IN SOLANUM FRUITS
and S. carolinense L.. Both species are annual or shortlived perennials common in agricultural and other disturbed landscapes throughout the southeastern United
States. In comparison with S. americanum, S. carolinense fruits are relatively low in sugar (3–4% vs. 5–
6% wet mass) and water content (80–90% vs. 90–95%
wet mass; M. Cipollini and D. Levey, unpublished
data). The large (ø10–20 mm in diameter), yellow
fruits of S. carolinense ripen in late fall and winter
(October through December) and are classified by
White (1989) as low-quality and mammal-dispersed
fruits. Martin et al. (1951) indicate that S. carolinense
is consumed by both birds and mammals. S. americanum ripens its small (5–9 mm in diameter), black
fruits in the summer and early fall (July through October) and is probably dispersed primarily by birds
(Martin et al. 1951), although small mammals also consume the fruits (Martin et al. 1951; M. Cipollini, personal observations). Both species have large numbers
of seeds, 40–170 seeds per fruit, but seeds of S. americanum (ø1.5–1.7 mm in diameter) are smaller than
those of S. carolinense (ø2.2–2.7 mm in diameter;
United States Department of Agriculture 1974). Our
alkaloid estimates (M. Cipollini, D. Levey, and R.
Bushway, unpublished data) indicate that unripe fruits
of both species contain 10–30 mg/g dry mass of total
glycoalkaloid, primarily a-solasonine and a-solamargine. However, ripe fruits of S. americanum are substantially lower in alkaloid content (,1 mg/g) than
those of S. carolinense (10–30 mg/g).
Fruits and seeds of S. americanum and S. carolinense
were collected from wild populations in Maryland and
Florida in 1991–1993. To provide sufficient fruit samples for feeding trials and chemical analyses, we propagated 20 seedlings derived from a single maternal
plant from each of 10 wild collections of each species
(200 total plants of each species). Plants were germinated and grown in a greenhouse, and then transplanted
to a tilled experimental field (Austin Cary Memorial
Forest, Alachua County, Florida).
Animal species
American Robins (N 5 14) were mist-netted in January, 1995 near Waldo, Florida. They are typically frugivorous at this time of year, and were maintained on
a fruit-based diet (Denslow et al. 1987). Northern Bobwhites (N 5 14), 10 wk old, were purchased from a
local breeder who regularly outbreeds captive stock
with wild populations. We fed them Purina Game Bird
Chow (Purina Mills, St. Louis, Missouri) and did not
conduct experiments with them until they were mature,
several months later. Deer mice (N 5 12) were obtained
from a captive population (D. Dewsbury, University of
Florida) that outbred with wild stock less than three
generations ago. They were fed LabDiet #5001 (Purina
Mills, St. Louis, Missouri). Opossums (N 5 9) were
trapped in December 1994 on the University of Florida
campus and maintained on Purina Cat Chow (Purina
785
Mills, St. Louis, Missouri). All animals were provided
with food and water ad libitum, except immediately
before a feeding trial, when food was removed for 8
h. They were kept on a 12 h:12 h L:D cycle (reversed
for mammals) and a constant temperature of 238C.
Feeding trials with birds were conducted under fluorescent light, whereas those with mammals were conducted during ‘‘dark’’ hours under red light. Our animal
handling and experimental protocol was reviewed and
approved by the University of Florida’s Institutional
Animal Care and Use Committee (Approval #1336).
These animal species were selected for several reasons: (1) they are reported consumers of Solanum fruits
(Martin et al. 1951), or have been observed feeding
upon Solanum fruits in the field (M. Cipollini, personal
observations); (2) a priori observations (M. Cipollini,
unpublished data) suggested that they would differ dramatically in their treatment of seeds (American Robins
and opossums are primarily seed dispersers; deer mice
and Northern Bobwhites are primarily seed predators);
(3) they were easily captured and maintained under
laboratory conditions; and (4) they could be trained
readily to consume ‘‘artificial fruit pulp’’ diets based
upon agar.
Extraction and purification of glycoalkaloids
We isolated a-solasonine and a-solamargine by column chromatography of crude glycoalkaloid extracts
from oven-dried samples of Solanum aculeatissimum
(provided by M. Weissenberg, Volcani Center, Israel),
S. carolinense, and S. americanum. Finely milled pulp
samples were pre-extracted with petroleum ether and
dichloromethane to remove fats, waxes, and pigments.
We then extracted the fruit pulp residue to exhaustion
with hot ethanol, and rotoevaporated the ethanol extract
to dryness. The dried ethanol extract was taken up with
1% acetic acid, the pH was shifted to ø11.5 using 30%
ammonia, and the extract was heated to 708C for 15
min. After the ammonified extract had cooled at 48C
overnight, we extracted the crude glycoalkaloid precipitate using chloroform. We rotoevaporated the crude
glycoalkaloid fraction to dryness, dissolved it in watersaturated l-butanol, and fractionated it on a 5 3 70 cm
chromatography column (neutral alumina). Fractions
(50 mL) eluted with water-saturated l-butanol were collected and monitored for purity using TLC (chloroform :
methanol : 1% ammonia (2:2:1); silica gel: Dragendorf’s
reagent). Pure fractions co-chromatographing with authentic a-solasonine and a-solamargine (authenticated
using TLC, HPLC, and FABMS by M. Weissenberg,
Volcani Center, Israel) were collected and rotoevaporated to dryness. Purity of the final extracts was verified
using HPLC (R. Bushway, unpublished data).
In all feeding experiments, we used citric acid to
adjust the pH of artificial fruits containing these glycoalkaloids to 5.7, the average pH of S. americanum
and S. carolinense fruits (M. Cipollini, unpublished
data). We do not know if pH affects toxicity of gly-
786
MARTIN L. CIPOLLINI AND DOUGLAS J. LEVEY
coalkaloids for vertebrates, but we felt that it was prudent to control for pH, because taste perception of Solanum glycoalkaloids is pH-dependent in humans
(spicy-hot at high pH, bitter at low pH; Zitnak 1979),
and glycoalkaloids have been noted to have pH-dependent effects in their antifungal and sterol-binding
activities (McKee 1959, Roddick et al. 1990, Fewell
and Roddick 1993).
Seed passage trials
To document differences in seed treatment among
our frugivore species, we fed three individuals of each
species a known quantity of S. americanum and, later,
S. carolinense fruits that had been bulk-collected from
many plants at our field site. Seed numbers in these
feedings were estimated from seed number : fruit mass
ratio determined on subsamples of these fruits. We collected all feces 24 h after fruit consumption, washed
them through a 0.5-mm mesh screen, and counted the
number of superficially intact seeds. Subsequent observations indicated that nearly 100% of seeds passed
through the gut of all studied animals within 24 h.
Additionally, tests using tetrazolium HCl (Hauser and
Morrison 1964) confirmed that seeds we classified as
intact contained viable embryos. We analyzed these
data using factorial ANOVA to determine the effect of
Animal Species, Fruit Species, and their interaction on
the arcsine-transformed proportion of seeds passed intact.
Real Fruit trials
We first addressed the General Toxicity vs. Directed
Toxicity Hypotheses via feeding trials using natural S.
americanum and S. carolinense fruits. Similar patterns
of consumption of these two species by seed predators
and seed dispersers would support the General Toxicity
Hypothesis, whereas different consumption patterns
would probably support the Directed Toxicity Hypothesis. We presented ø50 g of fresh fruit to eight individuals of each species. Trials lasted 15 min, after
which we recorded fruit mass consumed, Mc (N 5 6–
8 trials for each individual frugivore–fruit species combination). Trials were run in ‘‘sets,’’ so that within each
set, each frugivore was presented a random sequence
of eight fruit samples, four of which were S. americanum and four S. carolinense.
We analyzed preferences using a multivariate model,
repeated-measures ANOVA, with ln(Mc 1 1) as the
dependent variable and Fruit Species and Trial as within-factors variables (SuperANOVA; Abacus Concepts,
1989). For these and subsequent ANOVAs, the dependent variable (Mc) was ln-transformed prior to statistical analysis to reduce positive mean variance relationships. Additionally, for these and subsequent feeding trials, we ran at least three sets. If, after three sets,
any main effect was marginally significant (0.05 , P
# 0.10), we ran one or two additional sets and recalculated the statistics.
Ecology, Vol. 78, No. 3
This type of experimental design assumes that trials
within a set are random and independent, yet trials were
randomized sequentially for each frugivore within each
set. Consumption of a particular fruit type within any
trial might depend upon the fruit types experienced in
prior feeding trials. In order to reduce possible dependence among trials, we allowed a 15-min waiting period between successive feeding trials and randomly
presented fruit types to each frugivore. This assured us
that any trial with S. americanum had an equal probability of being preceded by a trial with S. americanum
or S. carolinense.
The type of short-term, single-choice feeding trial
approach we applied is a compromise between multiple-choice tests and long-term, single-choice tests.
Multiple-choice tests, where two or more food items
are presented simultaneously, may exacerbate preference differences and may also suffer from lack of independence (Peterson and Renaud 1989, Roa 1992).
Randomization of presentation order in short-term, single-choice trials presumably minimizes the problem of
dependence. Although relatively free from problems of
dependence, long-term, single-choice experiments
could underestimate preferences because animals may
eventually consume the less preferred foods, either as
hunger develops or after the more preferred foods have
been consumed.
Artificial fruit pulp trials
Real Fruit feeding trials were meant to assess fruit
preferences in the absence of confounding variables
present in the field (cf. Moermond and Denslow 1983,
Levey et al. 1984). Nevertheless, we lacked control
over many other fruit characteristics that may affect
fruit choice but were not of direct interest to us (e.g.,
color, size, seediness). To control such variables and,
thereby, focus on how nutrients and secondary metabolites influence fruit preference, we used artificial fruit
pulp diets. To reduce possible effects of habituation,
50–75% of the animals used for Real Fruit trials were
replaced by ‘‘naive’’ animals that we assumed had little
or no prior experience with these fruits.
Fruit Mimic trials.—The purpose of these trials was
to establish the chemical basis of preference patterns.
We mimicked the pulp chemistry of ripe and unripe
fruit in ‘‘nontoxic’’ (2) agars of each species, using
artificial components (carbohydrates, lipids, protein,
water, pH, and fiber; Table 1). Otherwise identical
‘‘toxic’’ (1) agars had the glycoalkaloids a-solasonine
(hereafter SA) and a-solamargine (hereafter SM) added
in proportions that reflected their concentrations in unripe or ripe fruits (Unripe (1) or Ripe (1) agars; Table
1). Concentrations of various constituents were determined by prior chemical analyses of unripe and ripe
fruits of each study species (M. Cipollini, D. Levey,
and R. Bushway, unpublished data). Feeding trails
were conducted and analyzed as for Real Fruit trials,
except that Agar Type was substituted for Fruit Species,
April 1997
EFFECTS OF GLYCOALKALOIDS IN SOLANUM FRUITS
787
TABLE 1. Chemical composition of artificial fruit agars used in this study. Data are mass (g) of each component used to
formulate 100 g of media (‘‘1’’ denotes presence of glycoalkaloids and ‘‘2’’ denotes their absence).
SUG†
CHO‡
LIP§
PRO\
FIB¶
SM#
SA††
Water
A) Fruit Mimic Trials
S. carolinense
Ripe (1)
Ripe (2)
Unripe (1)
Unripe (2)
3.75
3.75
2.94
2.94
1.50
1.50
0.70
0.70
0.75
0.75
0.70
0.70
1.05
1.05
1.12
1.12
7.35
7.35
7.90
7.98
0.264
0.000
0.255
0.000
0.000
0.000
0.008
0.000
85.00
85.00
86.00
86.00
S. americanum
Ripe (1)
Ripe (2)
Unripe (1)
Unripe (2)
5.39
5.39
1.12
1.12
0.70
0.70
0.32
0.32
0.35
0.35
0.28
0.28
0.56
0.56
0.48
0.48
0.00
0.00
1.64
1.64
0.005
0.000
0.041
0.000
0.003
0.000
0.047
0.000
93.00
93.00
96.00
96.00
4.00
4.00
4.00
4.00
1.00
1.00
1.00
1.00
0.58
0.58
0.58
0.58
0.50
0.50
0.50
0.50
4.00
4.00
4.00
4.00
0.000
0.100
0.200
0.000
0.200
0.100
0.000
0.000
90.00
90.00
90.00
90.00
C) Nutrient–Toxin trials
Low (1)
1.20
4.00
Average (1)
7.20
High (1)
4.00
Control (2)
0.30
1.00
3.20
1.00
0.15
0.58
0.85
0.58
0.15
0.50
0.85
0.50
8.20
4.00
0.00
4.00
0.100
0.100
0.100
0.000
0.100
0.100
0.100
0.000
90.00
90.00
90.00
90.00
Agar type
B) Interaction trials
SA (1)
SA 1 SM (1)
SM (1)
Control (2)
Note: All media were prepared using 2% agar solution held at 708C prior to the addition of alkaloid. Final pH was adjusted
to 5.7 with citric acid. Concentrations of various constituents were determined by prior chemical analysis of unripe and ripe
fruits of each study species (M. Cipollini, D. Levey, and R. Bushway, unpublished data).
† Sugars (glucose : fructose : sucrose, 2:2:1).
‡ Complex carbohydrates (starch : pectin, 3:1).
§ Lipids (peanut oil : corn oil, 1:1).
\ Protein (soy protein isolate).
¶ Fiber (milled pure cellulose fiber).
# a-solamargine.
†† a-solasonine.
and each set included two replicates of each Agar Type
3 Individual Frugivore combination. We tested all frugivores except opossums, which were excluded because of strong negative effects of glycoalkaloids and
the lack of influence of nutrient content on their response toward toxic (1) agars (see Results). When
Agar Type was a significant main effect, we made the
following planned comparisons: (1) vs. (2) agars
within each species for each ripeness state; ripe (1)
agars between species; and ripe (2) vs. unripe (2)
agars. The first comparison examined whether or not
glycoalkaloids influence consumption, the second comparison validated the Real Fruit trials, and the third
examined the effect of ripening, independent of glycoalkaloids, on consumption.
Interaction trials.—These trials were designed to determine whether the two alkaloids (SA and SM) differed in their activity toward the frugivores and to determine whether there was an interaction (synergism)
in their effects at levels typical of ripe S. carolinense
fruits. Such synergisms are important to explore, since
both SA and SM are commonly present in Solanum
fruits and synergism between these glycoalkaloids is
known to occur (e.g., Roddick et al. 1990). We formulated a nontoxic (2) agar that approximated average
levels of carbohydrate, lipid, protein, water, pH, and
fiber (Table 1). Toxic (1) agars had SA and SM added
at overall levels of 2% dry mass, a concentration typical
of S. carolinense and other toxic ripe Solanum species
(M. Cipollini, D. Levey, and R. Bushway, unpublished
data). The SA:SM ratio was varied among the three
(1) agars: (1) SA only; (2) SA 1 SM, 1:1; and (3) SM
only (Table 1). We conducted Interaction trials for all
four animal species and analyzed the data as described
for Fruit Mimic trials. Planned comparisons were: ‘‘SA
only’’ and ‘‘SM only’’ vs. ‘‘SA 1 SM,’’ and ‘‘SA
only,’’ ‘‘SM only,’’ and ‘‘SA 1 SM’’ vs. ‘‘Control.’’
The first contrast tests for interaction: whether or not
the average consumption of SA and SM agars alone
differs from consumption of the agar containing both
SA and SM. The second contrast examines the average
influence of glycoalkaloids on consumption.
Nutrient–Toxin Titration trials.—These trials more
specifically addressed the Nutrient–Toxin Trade-off vs.
the Removal Rate Hypotheses by focusing on frugivore
response to variation in nutrient levels while alkaloid
levels were held constant. We formulated a nontoxic
(2) agar identical to that used in the Interaction trials,
and three toxic (1) agars containing 2% dry mass glycoalkaloid (1:1, SA:SM; Table 1). We varied the overall
MARTIN L. CIPOLLINI AND DOUGLAS J. LEVEY
788
Ecology, Vol. 78, No. 3
RESULTS
Seed Passage trials
Based upon the proportion of seeds they passed intact, we classified American Robins and opossums as
seed dispersers, and Northern Bobwhites and deer mice
as seed predators. The latter two species destroyed 85–
95% of seeds they consumed, whereas the former destroyed few or none (Fig. 1). This classification is not
meant to imply that Northern Bobwhites and deer mice
are strictly seed predators and ineffective dispersers of
the viable seeds they defecate, only that their treatment
of seeds is generally less beneficial to Solanum plants
than treatment by American Robins and opossums. Factorial ANOVA results (Table 2) support this dichotomy,
with a significant Animal Species effect, but nonsignificant Fruit Species and interaction effects.
Real Fruit trials
FIG. 1. Results of Seed Passage trials. Data are means 6
1 SE for the proportion of seeds ingested that were passed in
viable condition (N 5 3 individuals of each animal species).
Identical letters above vertical bars indicate animal species
that did not differ in their seed passage rate for a particular
fruit species, based upon one-way ANOVA (P # 0.05).
nutrient concentration in the three (1) agars as follows:
Low (1), 70% reduction in overall nutrient content;
Average (1), nutrient concentration equal to the control; and High (1), a 70% increase in overall nutrient
content (Table 1). Different amounts of fiber compensated for varying levels of nutrients. Final nutrient concentrations fell within the range reported for temperate
fleshy fruits (Stiles 1980, Stiles and White 1982, White
1989). We conducted Nutrient–Toxin Titration trials for
all four animal species and analyzed the data as described for Fruit Mimic trials. Planned comparisons
were: Low (1) vs. Average (1) and High (1) vs. Average (1), which tested whether nutrient levels below
or above average influence consumption when glycoalkaloid content is held constant.
Consumption rates of S. carolinense were not significantly elevated in sets run with open fruits, and this
treatment effect was thus eliminated from the ANOVA
models. Northern Bobwhites, deer mice, and opossums
strongly preferred S. americanum over S. carolinense
fruits; American Robins showed a similar, but marginally significant (P 5 0.085), preference (Fig. 2). Consumption rates were 4–11 times greater for S. americanum than for S. carolinense. Consumption rates varied significantly among trials for all species (Table 3),
but not in a consistent manner for any species. The
Trial 3 Fruit Species interaction was not significant for
any species. For all species except American Robin,
these results demonstrate that fruit species have a
strong and consistent influence on consumption.
Artificial fruit pulp trials
Fruit Mimic trials.—Agar type strongly influenced
consumption rate for all three species (P , 0.001; Table
4). Trial and Agar Type 3 Trial terms were not significant for Northern Bobwhites and deer mice, but
were significant for American Robins. Robins were
much more variable than the other species in their feeding pattern. Individuals varied in their consumption
rates, and consumption rates varied among sets. How-
TABLE 2. Results of factorial ANOVA for Seed Passage trials. The dependent variable was
the arcsine-transformed proportion of seeds passed intact in feces.
df
3
11.184
Fruit Species
(S. carolinense vs. S. americanum)
1
0.008
0.31
Animal 3 Fruit Species
3
0.148
1.94
25
0.634
Error
**** P # 0.0001.
Type III
F
Source
Animal species
(American Robin vs. opossum vs.
Northern Bobwhite vs. deer mouse)
SS
147.02****
EFFECTS OF GLYCOALKALOIDS IN SOLANUM FRUITS
April 1997
FIG. 2. Results of Real Fruit trials. Data are means 6 1
for the relative amount of fruit mass consumed per trial
(i.e., mass of a particular fruit consumed divided by the total
mass of all fruits consumed; N 5 8 individuals of each animal
species). Matching lowercase letters indicate nonsignificant
differences between plant species (P . 0.05; planned contrasts). Where the same letter appears in upper and lowercase,
the difference is significant (P , 0.05).
SE
ever, an examination of the temporal sequence of preference patterns for robins revealed no evidence that
choices expressed in early trials were consistently different from those expressed in later trials.
Planned contrasts of means demonstrated that Northern Bobwhites and deer mice generally consumed significantly lower amounts of agars containing toxins
(Fig. 3). For Northern Bobwhites, three of four comparisons between agars identical except for the presence or absence of glycoalkaloids were significant at
P , 0.0001, and the remaining comparison at P 5 0.08
(Fig. 3). Deer mice showed the same pattern. Both
species failed to distinguish between agars that mimicked ripe S. americanum with and without glycoal-
789
kaloids. Robins did not display any significant preference for agars without glycoalkaloids ( P 5 0.55 6
0.35, mean 6 1 SE, N 5 4 comparisons); they appear
less sensitive to them. For each fruit species and ripeness state, however, robins consistently consumed (1)
agars at lower levels than (2) agars (Fig. 3).
All three species significantly preferred agars mimicking ripe S. americanum fruits over those mimicking
ripe S. carolinense (ripe ‘‘1’’ agars: all P , 0.002,
planned comparisons; Fig. 3). These results confirm
those of the Real Fruit trials, in which S. americanum
was strongly preferred by Northern Bobwhites and deer
mice, and slightly preferred by American Robins.
Planned comparisons of ripe S. americanum and S. carolinense agars lacking glycoalkaloids (‘‘ 2’’ agars)
were insignificant for Northern Bobwhites and deer
mice (P 5 0.09 and 0.48, respectively) and significant
for American Robins (P 5 0.0.16; Fig. 3). These results
suggest that the higher concentrations of glycoalkaloids
in ripe S. carolinense fruits were more important in
determining preference for S. americanum than were
differences in nutrient content, since two of the three
animal species were able to distinguish between the
two fruit species only when glycoalkaloids were present.
Interaction trials.—The main effect of Agar Type
was highly significant for all four animal species (Table
5). In every case, this was due to significantly higher
consumption of the nontoxic control agar, relative to
the three toxic agars (all P , 0.001, planned comparisons; Fig. 4). Measured consumption (in grams) was
4–14 times greater for control agars than for the other
agars. Consistent with results of the Fruit Mimic trials,
American Robins displayed the lowest difference between consumption of these agar types, indicating that
they were least sensitive to the presence of glycoalkaloids. Opossums were the most sensitive.
Set and Set 3 Agar Type terms were significant for
opossums and Northern Bobwhites. For Northern Bobwhites, consumption of control agars consistently increased from one set to the next. For opossums, consumption of control agars varied widely among sets,
with no discernible trend. In both cases, the significant
interaction term was due to the large variation in con-
TABLE 3. Results of repeated-measures ANOVA for Real Fruit feeding trials. The dependent variable was the ln-transformed
fruit mass eaten per trial. Results are presented separately for each species. Data are F values based upon Type III sums
of squares.
American Robin
Source
F
Fruit species
(S. carolinense vs.
S. americanum)
Set (repeated)
Fruit Species 3 Set
Opossum
df
F
4.0
1
1.7*
15
0.9
15
Northern Bobwhite
df
F
105.6****
1
3.5***
11
1.2
11
* P # 0.05; ** P # 0.01; *** P # 0.001; **** P # 0.0001.
Deer mouse
df
F
96.8****
1
29.3***
1
3.0***
15
2.4**
14
1.4
15
0.8
14
df
MARTIN L. CIPOLLINI AND DOUGLAS J. LEVEY
790
Ecology, Vol. 78, No. 3
TABLE 4. Results of repeated-measures ANOVA for Fruit Mimic feeding trials using agars
with (1) or without (2) alkaloid. The dependent variable was the ln-transformed agar mass
eaten per trial. Results are presented separately for each species. Data are F values based
upon Type III sums of squares.
American Robin
Northern Bobwhite
Deer mouse
F
df
F
df
F
Agar Type†
4.3***
7
27.5****
7
13.4****
Set (repeated)
3.1**
9
0.8
5
1.2
5
Agar Type 3 Set
2.0****
63
0.9
35
0.7
35
Source
df
7
* P # 0.05; ** P # 0.01; *** P # 0.001; **** P # 0.0001.
† S. carolinense ripe (1) vs. S. carolinense ripe (2) vs. S. carolinense unripe (1) vs. S.
carolinense unripe (2) vs. S. americanum ripe (1) vs. S. americanum ripe (2) vs. S. americanum
unripe (1) vs. S. americanum unripe (2).
sumption of control agars relative to consistently low
and relatively equal consumption of the other agar
types. Although they differed in magnitude of response,
both species that we classified as seed dispersers (robins and opossums) were deterred by glycoalkaloid at
levels found in ripe fruits. We found no evidence that
seed predators (Northern Bobwhites and deer mice)
were targeted specifically by these compounds.
We also found no evidence of synergism between
the two glycoalkaloids in their deterrent effects. In particular, consumption of the SA 1 SM agar did not differ
from the average consumption of the SA-only and
SM-only agars (all P . 0.5, planned comparisons; Fig.
4).
Nutrient–Toxin Titration trials.—If nutrient concentration can compensate for the deterrence produced by
glycoalkaloid concentrations typical of ripe S. carolinense fruits, we would expect to find strong differences
in consumption of toxic (1) agars that differ dramatically in overall nutrient content. Although Agar Type
was by far the most important determinant of agar consumption for all species (all P , 0.0001; Table 6) in
Nutrient–Toxin Titration trials, most variation in consumption among agar types was due to strong preferences for control (2) agars over (1) agars containing
glycoalkaloids and varying amounts of nutrients (all P
, 0.0001, planned comparisons; Fig. 5). As in the Interaction trials, opossums were most sensitive to presence of glycoalkaloids; they consumed ø27 times more
control agar than the most preferred toxic agar. Deer
mice were least sensitive (consuming two times more
control agar), followed by American Robins (2.6 times
more control agar) and Northern Bobwhites (3.7 times).
Average consumption rate of (1) agars containing
glycoalkaloids was ranked identically by all species:
High nutrient content . Average nutrient content .
Low nutrient content. However, with one exception
(High (1) vs. Average (1) for deer mice; P , 0.05),
differences in consumption were not significant in pairwise tests (High vs. Average and Low vs. Average;
planned comparisons.)
Thus, only deer mice responded significantly to vari-
ation in nutrient composition of toxic agars, and not
enough to fully compensate for the negative effect of
glycoalkaloid (Fig. 5). For all species, we found no
significant differences between Low (1) and Average
(1) agars. Feeding rates on High (1) agars were about
twice the feeding rates on Low (1) agars. Nevertheless,
feeding rates for all species on High (1) agars were
still ,50% of those on nontoxic (2) control agars.
Given the large magnitude of nutrient variation in the
toxic agars tested, ranging from a 70% reduction in
nutrient content in Low (1) to a 70% increase in High
(1), these results further support the conclusion that
glycoalkaloid content is a much more important determinant of agar consumption than is nutrient content.
Set effects were significant for opossums, deer mice,
and Northern Bobwhites (Table 6). As in the Interaction
trials, Northern Bobwhites increased their consumption
of control agars during the course of the sets. Opossums
and deer mice showed high but inconsistent differences
among sets in consumption of control agars. Consumption of other agars did not vary in a consistent
manner from one set to the next, except for Northern
Bobwhites, which greatly increased their consumption
of the high-nutrient agar in two of the last three sets.
Largely as a result, Northern Bobwhites had a significant Agar Type 3 Set interaction term (P , 0.0001;
Table 6).
DISCUSSION
Overview of results
All four animal species, both seed predators and seed
dispersers, were deterred by glycoalkaloid levels and
combinations typical of ripe Solanum fruits. Although
American Robins seemed to be less deterred by glycoalkaloids than were the other species in some trials
(e.g., Real Fruit and Fruit Mimic trials), results of other
trials (e.g., Interaction and Nutrient–Toxin Titration trials) showed strong negative effects of glycoalkaloids
upon robins. These results lead us to reject the Directed
Toxicity Hypothesis. Deterrent effects of glycoalkaloids were so strong that they generally overrode the
April 1997
EFFECTS OF GLYCOALKALOIDS IN SOLANUM FRUITS
791
effects of nutrient content in preference trials; even
highly rewarding artificial pulp was usually rejected if
glycoalkaloids were present. Thus, we also reject the
Nutrient–Toxin Titration Hypothesis. Our general conclusion is that glycoalkaloids in ripe Solanum fruits are
best explained by the General Toxicity and Removal
Rate Hypotheses.
Real fruit and artificial fruit preferences
The frugivores we tested generally consumed Solanum americanum fruits at rates 4–10 times higher than
they consumed S. carolinense fruits. More important,
an animal we classified as a potential seed disperser
(opossum) was as strongly deterred from feeding on
the high-alkaloid S. carolinense as were those animals
we classified as seed predators (Northern Bobwhite,
deer mouse). Although American Robins were not significantly deterred from feeding on S. carolinense, they
fed at a rate about four times lower for S. carolinense
relative to S. americanum in these trials. It seems that
our experimental design was not sufficient to pick up
this preference by robins for S. americanum. When the
data were analyzed using an alternative factorial model
that included effects of individual frugivores, the preference of American Robins for S. americanum was significant (results not shown). These results argue against
the Directed Toxicity Hypothesis, although as discussed previously, aspects of fruit physical and chemical design other than fruit toxicity may have influenced
the fruit preferences of these organisms. Thus, we used
feeding trials with artificial fruits to isolate nutrient and
toxin effects.
In general, results of Fruit Mimic trials supported
the hypothesis that both Northern Bobwhites and deer
mice were affected negatively by glycoalkaloids. Effects of nutrient level on consumption were generally
small, except for American Robins, which responded
negatively to low-nutrient agars mimicking S. carolinense fruit pulp. Alkaloids in S. carolinense toxic agars
were nearly 100% SA, whereas S. americanum toxic
agars contained ø50% each of SA and SM. Unripe S.
americanum toxic (1) agars (at 0.08% of wet mass)
had an effect similar to that of S. carolinense toxic (1)
agars (ø0.26% of wet mass), possibly the result of
chemical synergism. Such synergism has been demonstrated for these two alkaloids in their effects upon
lipid membrane disruption (Roddick et al. 1990), albeit
at concentrations nearly five times higher than those
employed in our tests. But, because the results of our
←
FIG. 3. Results of Fruit Mimic trials. Data are means 6
1 SE for the ln-transformed mass of agar consumed per trial
(N 5 8 individuals per animal species). Letters indicate
planned comparisons that apply within species only (see Fig.
2). For any particular fruit species–fruit state combination,
(2) indicates no added alkaloid, whereas (1) indicates addition of alkaloid. For agar compositions, refer to Table 1.
MARTIN L. CIPOLLINI AND DOUGLAS J. LEVEY
792
Ecology, Vol. 78, No. 3
TABLE 5. Results of repeated-measures ANOVA for Interaction feeding trials. The dependent
variable was the ln-transformed agar mass eaten per trial. Results are presented separately
for each species. Data are F values based upon Type III sums of squares.
American Robin
Source
Agar type†
Northern
Bobwhite
Opossum
Deer mouse
F
df
F
df
F
df
F
df
25.2****
3
93.8****
3
16.4****
3
43.5****
3
Set (repeated)
1.1
7
8.9****
5
4.2**
5
1.1
5
Agar Type 3 Set
1.4
21
4.9****
15
2.5**
15
0.9
15
* P # 0.05; ** P # 0.01; *** P # 0.001; **** P # 0.0001.
† ‘‘SA only’’ vs. ‘‘SA 1 SM’’ vs. ‘‘SM only’’ vs. ‘‘Control’’.
Interaction trials provided no support for synergism
with respect to the deterrence by these compounds, we
do not think that synergism explains the similarity in
the consumption rate of toxic (1) unripe S. americanum
and S. carolinense agars. For all species, agars containing 1:1 mixtures of SA and SM were consumed at
rates similar to those of agars containing only one of
the two compounds. Although sample sizes were too
FIG. 4. Results of Interaction trials. Data are means 6 1 SE for the relative amount of artificial fruit agar consumed per
trial (i.e., mass of a particular agar consumed divided by the total mass of all agars consumed; N 5 8 individuals of each
animal species). Letters indicate planned comparisons as in Figs. 2 and 3, except that underlined letters define members of
a group whose mean response was compared to the matching letter not underlined. For agar compositions, refer to Table 1.
EFFECTS OF GLYCOALKALOIDS IN SOLANUM FRUITS
April 1997
793
TABLE 6. Results of repeated-measures ANOVA for Nutrient–Toxin Titration feeding trials. The dependent variable was
the ln-transformed agar mass eaten per trial. Results are presented separately for each species. Data are F values based
upon Type III sums of squares.
American Robin
Source
Agar Type†
F
36.6****
Set (repeated)
0.7
Agar Type 3 Set
2.0**
Opossum
Northern Bobwhite
df
F
df
F
3
80.3****
df
Deer mouse
F
df
3
34.8****
3
35.8****
3
7
2.9*
5
7.3****
7
6.9***
5
21
1.5
15
3.1****
21
1.5
15
* P # 0.05; ** P # 0.01; *** P # 0.001; **** P # 0.0001.
† ‘‘Low Nutrient (1)’’ vs. ‘‘Average Nutrient (1)’’ vs. ‘‘High Nutrient (1)’’ vs. ‘‘Control’’.
small for statistical tests, there were no obvious differences between the responses of naive and experienced animals in successive sets of feeding trials. These
observations suggested that the preferences exhibited
by these animals were either innate or learned rapidly
upon initial presentation of choices. Because results of
our repeated feeding trials were consistent, we have
assumed that learning was not an important component
of the choices made by our frugivores. Nevertheless,
we recognize that feeding deterrence might be learned
in animals as a result of a single toxic (nonfatal) dose
of a chemical (e.g., Robbins 1978). If this were true
for our feeding trials, our conclusions would remain
basically unaltered. Whether innate or rapidly learned,
aversions to these toxins greatly affected choices made
by these frugivores.
We believe that glycoalkaloids in our artificial fruit
pulp closely mimicked the effects of naturally occur-
FIG. 5. Results of Nutrient–Toxin Titration trials. Data are means 6 1 SE for the relative amount of artificial fruit agar
consumed per trial (refer to Fig. 4). Letters indicate planned comparisons as in Fig. 4. For agar compositions, refer to
Table 1.
794
MARTIN L. CIPOLLINI AND DOUGLAS J. LEVEY
ring glycoalkaloids in fruit, and that these glycoalkaloids are a primary determinant of fruit acceptability
to frugivores. In support, we note that these animals
preferred S. americanum over S. carolinense fruits by
approximately the same ratio that they preferred agars
mimicking S. americanum fruits over those mimicking
S. carolinense fruits. Fruits of native or naturalized
Solanum species similar to S. americanum (e.g., S. luteum Mill., S. nigrum L., S. sarrachoides Sendt. in
Mart., and S. ptychanthum Dun. ex DC.) are similar in
alkaloid profile to fruits of S. americanum (Carle 1981,
Tamboia et al. 1996). These fruits are eaten by captive
birds and mammals with as much relish as are fruits
of S. americanum (Barnea et al. 1990, Tamboia et al.
1996; M. Cipollini, unpublished data). On the other
hand, several exotic species of large, yellow-fruited
Solanum are similar to S. carolinense in that they are
high in glycoalkaloids in the ripe state (e.g., S. viarum
Dun., S. aculeatissimum Jacq., and S. mammosum L.).
Although large domestic animals such as cattle seem
to play some role in the dispersal of these introduced
weeds (e.g., Mullahey and Colvin 1993), wild animals
tend to avoid these fruits, which persist for long periods
of time on plants without being removed (M. Cipollini,
personal observations). As with S. carolinense, such
‘‘toxic’’ fruits tend to be consumed and dispersed in
Florida primarily by mammals such as whitetail deer
(Odocoileus virginianus), raccoons (Procyon lotor),
Virginia opossums and feral hogs (Sus scrofa) (White
1989; Mullahey and Colvin 1993; M. Cipollini, personal observations). Taken together, these observations
suggest that the results of our comparison of S. americanum and S. carolinense are probably generalizable
to a broader array of Solanum species than we were
able to compare experimentally.
Although robins, which are potential dispersers of
Solanum fruits, seemed less affected by glycoalkaloids,
oppossums, our other potential dispersers, were most
affected. Additionally, we have tested Cedar Waxwings
(Bombycilla cedrorum), also potential dispersal agents,
and found them to be equally deterred as were the
animals tested in this study (D. J. Levey and M. L.
Cipollini, unpublished data). Evidence that both seed
predators and seed dispersers were affected negatively
by toxins in Fruit Mimic trials provides direct support
for the General Toxicity Hypothesis. These results were
further supported by strong negative effects of glycoalkaloids on feeding rates of all species in both Interaction and Nutrient–Toxin Titration trials, regardless
of the identity of the alkaloids.
The generally small effects of nutrient composition
seen in both Fruit Mimic trials and Nutrient–Toxin Titration trials suggest that, although nutrient composition may play a role in influencing consumption, the
effects of glycoalkaloids are overriding. We found little
evidence that increased nutrient content can compensate for toxin content at the levels found in toxic Solanum fruits such as S. carolinense. Thus, our results
Ecology, Vol. 78, No. 3
are not consistent with the Nutrient–Toxin Titration
Hypothesis, but are consistent with the Removal Rate
Hypothesis. That is, fruits high in nutrient content, otherwise profitable (e.g., low fiber content), or quickly
removed by dispersers should contain relatively low
amounts of toxins. S. americanum fruits were generally
preferred over those of S. carolinense in Real Fruit
trials, and ripe S. americanum fruit-mimicking agars
were preferred by one of the frugivores tested (American Robins). Our field observations over four years
indicate very low and sporadic dispersal rates for S.
carolinense (most fruits generally fall to the ground
and rot), but relatively consistent removal rates for S.
americanum. As predicted, S. americanum fruits have
negligible levels of glycoalkaloid in ripe fruits (generally ,0.2% of dry mass), whereas S. carolinense
fruits have high levels (1–3%) in ripe fruit.
Selection for persistence in temperate-zone fruits
Different fruiting phenologies and consequent requirements for persistence may ultimately explain differences in alkaloid content of S. americanum and S.
carolinense. In temperate North America, S. americanum and other members of the S. nigrum complex
generally ripen fruits in the summer and early fall,
when dispersal rates by resident and migrant frugivorous birds are high (Thompson and Willson 1979). Conversely, S. carolinense fruits ripen in late fall and may
persist throughout the winter into early spring (M. Cipollini, personal observation). Dispersal by frugivorous birds and mammals may be very unpredictable
and sporadic for fruits at this time (White and Stiles
1990). Thus, differences in the removal rate of these
species in the field may reflect extrinsic differences in
the temporal dynamics of the frugivore community.
Like several other North American plants (e.g., Ilex
opaca Ait., Vaccinium macrocarpon Ait., Rhus typhina
L., and Juniperus virginiana L.; White 1989), S. carolinense fruits may have to persist for long periods of
time, ‘‘waiting’’ for visitation by dispersal agents. During this time, they presumably require protection
against damaging agents such as fungi and insect pests.
As previously discussed, glycoalkaloids are known to
be toxic and to act as feeding deterrents for generalist
invertebrate herbivores, and they are known to have
strong antifungal effects (McKee 1959, Allen and Kuc
1968, Allen 1970, Roddick 1987, Cipollini and Levey
1997). Thus, retaining such general toxins may be selectively advantageous by preserving fruits during periods of unpredictable and/or sporadic dispersal.
A similar pattern has been found for at least two sets
of species: (1) temperate ericaceous plants (Cipollini
and Stiles 1992, 1993), and (2) temperate Ilex species
(Gargiullo and Stiles 1991). In both cases, relative to
species that ripen in late fall and winter, summer or
early-fall species were removed at higher rates by seed
dispersers, tended to be preferred by frugivorous birds,
and contained lower amounts of (or less active) sec-
April 1997
EFFECTS OF GLYCOALKALOIDS IN SOLANUM FRUITS
ondary metabolites. Feeding trials using artificial fruit
pulp agars mimicking ericaceous species demonstrated
that preferences for summer species were based on both
nutrient and secondary metabolite content (Cipollini
and Stiles 1993).
Alternative hypotheses
Although our experiments suggest that secondary
metabolites in ripe fruits represent a trade-off between
defense against pests/pathogens and palatability for
dispersal agents, other explanations are not precluded.
For instance, Murray et al. (1994) suggest that chemicals within the fruits of another solanaceous plant
(Witheringia solanceae Remy) have a laxative effect
and enhance seed passage rates in Black-faced Solitaires (Myadestes melanops) feeding on these fruits.
Rapid seed passage is correlated with lower seed damage during gut passage. We note that one of the first
symptoms of Solanum glycoalkaloid toxicity is irritation of the gut lining and diarrhea (Van Gelder 1990).
Also, Sorensen (1983), Barnea et al. (1993), and Izhaki
and Safriel (1989) suggest that fruit pulp chemicals
may be present within ripe fruits as a means of inducing
frugivores to leave fruiting plants early in a feeding
bout, enhancing dispersal of seeds away from the parent
plant. In S. carolinense, reproduction within a given
habitat is strongly dominated by clonal growth via rhizomes (Nichols et al. 1991). The occurrence of these
plants only in highly disturbed areas such as farm fields
and riverbeds suggests that dispersal into and out of
these ephemeral habitats may be extremely important.
Thus, glycoalkaloids of ripe fruits may play a role in
inducing consumers to seek alternative food sources
after consuming one or a few fruits.
Nonadaptive explanations for the presence of glycoalkaloids and other secondary metabolites in ripe
fruits should also be considered. Immature fruits commonly accumulate secondary metabolites, many of
which decrease substantially with ripening (Goldstein
and Swain 1963, Dement and Mooney 1974, Eltayeb
and Roddick 1984a, b). Plants often recover these
chemicals from senescent tissues, but may vary in their
ability to do so. Interspecific variation in the secondary
metabolites of ripe fruits might, thus, be a nonadaptive
result of differing patterns of plant senescence. We note
that, for our study species, this explanation is unlikely.
Fruit production is indeterminate, with continuous
asynchronous ripening, in the ‘‘nontoxic’’ S. americanum, whereas it is determinate, with synchronous,
postsenescent ripening in the ‘‘toxic’’ S. carolinense.
In conclusion, we have demonstrated that secondary
metabolites of ripe fleshy fruits may strongly affect
fruit choice by vertebrates. The magnitude of their effects, overriding large differences in nutrient content,
may help to explain why previous studies on the influence of fruit characters on fruit choice have yielded
inconsistent or inconclusive results (e.g., Sorensen
1983, Johnson et al. 1985). If fruit secondary metab-
795
olites are not considered in such studies, fruit preferences based on nutrient content may be difficult to
detect. More generally, we hope that our results spur
further research into the implications of secondary metabolites of fleshy fruits. Our results suggest that studies
of interactions between fruiting plants and seed dispersers
need to include the influences of other frugivores, including seed predators, fruit pests, and pathogens (cf.
Herrera 1984, 1989, Borowicz 1988, Buchholz and
Levey 1990, Cipollini and Stiles 1993). We suggest
that if such frugivores did not impinge upon the fruit–
seed disperser mutualism, patterns of fruit ripening and
secondary metabolite content would be vastly different,
as would interactions among fruiting plants and their
seed dispersers.
ACKNOWLEDGMENTS
We thank R. Bushway for HPLC analysis of glycoalkaloids,
L. Baggett, I. Cipollini, M. E. Cipollini, S. Cipollini, W. Deckard, P. Rey Zamora, W. Silva, T. Tamboia, and G. Williams
for field and lab assistance, C. Restrepo for statistical advice,
C. Augspurger, C. Herrera, W. Karasov, and T. Stiles for
helpful comments and criticism of the hypotheses and methods of this research, D. Schultz and S. Brooker for logistical
assistance at Austin Cary Memorial Forest, M. Weissenberg
for glycoalkaloid standards and isolation methods, C. Sacchi,
D. Whigham, J. O’Niell, M. Gargiullo, and R. Frank for fruit
samples, Mr. and Mrs. Garrett for permission to mist-net birds
on their property, J. Davis for assistance with animal care
facilities, D. Dewsbury for providing field mice, M. Sunquist
for use of live traps, L. Brower and B. McNab for use of
animal cages, and M. Debussche and an anonymous reviewer
for helpful comments and criticism. This work was supported
by NSF grant DEB-9207920 and an NSF Research Experiences for Undergraduates Supplemental Grant.
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