Int. J. Plant Sci. 164(3 Suppl.):S93–S102. 2003.

䉷 2003 by The University of Chicago. All rights reserved.

1058-5893/2003/16403S-0007$15.00

THE EVOLUTION OF FUNCTION IN PLANT SECONDARY METABOLITES

Nina Theis and Manuel Lerdau1
Ecology and Evolution Department, State University of New York, Stony Brook, New York 11794-5245, U.S.A.

For the past 40 years, the ecology and evolution of plant secondary metabolites have been major foci of
investigation. We use one class of secondary metabolites, the terpenes, as a case study for exploring the factors
regulating the evolution of metabolite function. Evolution of function can occur as a result of change at any
of several scales of biological organization. Changes in gene sequence and/or genetic architecture underlie
several important evolutionary changes in function, and changes in gene regulation that alter terpene quantities
also are linked with functional shifts. In addition, changes in the spatial distribution of terpenes within plants
and in the structures used for terpene storage can be involved with functional shifts. Finally, as volatile
compounds, terpenes play important roles as signaling compounds, and evolution of function can occur through
changes in organisms that receive signals as well as the organisms that send them. In terpenes, significant
changes in function have occurred as insects have learned to use terpenes as attractant or deterrent cues. A
complete understanding of the evolution of function in secondary metabolites requires studying the regulation
of function across all of these scales.

Keywords: secondary metabolite, terpene, terpenoid, allocation, chemical defense, chemical ecology.

Introduction
Nowhere in the plant kingdom do ecology, evolution, and
human affairs come together as intricately as in phenomena
involving secondary metabolites. (Secondary metabolites are
those compounds produced by plants that are not directly es-
sential for basic photosynthetic or respiratory metabolism;
such compounds are known as primary metabolites.) The im-
portance of these compounds is apparent in some of the earliest
annals of human history. For example, Papaver somniferum,
the original source of morphine and codeine, was known to
the Sumerians in 4000 BCE as hul gil (joy-plant). These two
alkaloids, so important in human affairs for many millennia,
probably evolved within the genus Papaver originally as de-
fensive compounds against herbivores. As a testament to the
importance of secondary metabolites, P. somniferum was one
the first plants to be cultivated for a reason other than its
caloric or nutritive value. Sadly, its importance has only in-
creased over time.
Organic chemists of the nineteenth century were among the
first to recognize and study the diversity of plant secondary
metabolites and to analyze them both in terms of structure
and function. Chemical modification of morphine, obtained
from P. somniferum, was one of the Bayer Company’s first
great commercial successes. The German organic chemists de-
veloped techniques to acetylate morphine and produce what
they claimed at the time to be a nonaddictive version of the
drug. Soon, however, the company realized that their new
product, sold under the trade name Heroin, was vastly more
addictive. The economies of several developing nations in Asia
still depend on the acetylation of this plant secondary metab-
1
Author for correspondence; e-mail manuel.lerdau@sunysb.edu.
Manuscript received September 2002; revised manuscript received January 2003.
olite, and tens of thousands of lives each year are destroyed
by its commercial importance. In another striking example of
profitable secondary metabolites, the Bayer Company later ap-
plied similar acetylation techniques to salicylic acid, derived
originally from the bark of willows, Salix, to create aspirin.
The modern appreciation of these secondary metabolites
from ecological and evolutionary perspectives had its spread
outside of the plant-insect ecology community with Fraenkel’s
1959 article in Science, “Raison d’Être of Secondary Plant
Substances.” In this article he postulated that the overwhelm-
ing array of chemicals found in plants were more than simply
by-products of primary metabolism that served as waste prod-
ucts for these organisms that lacked excretory systems. By the
time of Fraenkel’s article, the dust had largely settled on the
question of whether nonnutritive factors such as attractants
or repellents played a major role in insect host preference.
Fraenkel, Dethier (1954), and their colleagues had provided
overwhelming evidence that insects paid attention to these sec-
ondary substances in making their choices about what plants
to eat.
Within 1 year of Fraenkel’s 1959 publication, extensive re-
view articles in both the plant physiological and entomological
literature addressed the topic of the functions of secondary
substances (Neish 1960; Thorsteinson 1960). Ecologists and
evolutionary biologists soon took up the cudgels, and the past
40 years have seen the development, testing, refinement, and,
in some cases, rejection of models that describe the ecology
and evolution of these “secondary” substances. With the func-
tional roles of these compounds broadly acknowledged, several
challenges presented themselves to scientists interested in their
ecological and evolutionary dynamics. Most obviously, the de-
tails of the processes underlying the interactions between each
of these compounds and their target pest or pathogen became

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S94 INTERNATIONAL JOURNAL OF PLANT SCIENCES
(and remain) one of the most vibrant topics in ecological and
physiological research.
Two features of these chemicals and the interactions between
the plants that produce them and their respective pests raised
questions that continue today to motivate ecologists, evolu-
tionists, agriculturalists, and legions of graduate students. First
of all, the staggering diversity of “secondary” chemicals found
in plants, even in closely related taxa, immediately raised ques-
tions regarding the origin and maintenance of this diversity.
Second, the role of these chemicals in modulating plant-pest
interactions and the prevalence of host specificity among phy-
tophagous insects are poorly understood but essential issues
for ecological and evolutionary studies as well as for applied
questions in agriculture.
Evolutionary and ecological studies of plant secondary me-
tabolites have been closely linked because of the early recog-
nition that many of these compounds played distinct ecological
roles while also showing phylogenetic affinities, such that de-
tails of the distribution of chemical structures among taxa
could not be explained without considering evolutionary re-
lationships. However, it soon became apparent that phyloge-
netic relationships among plants were not sufficient to predict
functional roles of secondary compounds. For example, chem-
ically similar secondary metabolites such as terpenes can serve
a range of functions, including herbivore deterrence, fungal
toxicity, and pollinator attraction; they can also function as
solvents for higher molecular mass compounds that would
otherwise solidify and clog transport systems in plants.
Ecological and evolutionary models have focused on the
origin and maintenance of plant chemical diversity and on the
regulation of chemical concentrations within plants. There has
been less effort to date on formal models of the evolution of
secondary metabolite function and of how function may
change over time, though some researchers have examined
these questions on the scale of particular plant clades (Arm-
bruster et al. 1997). Rather than attempt to cover the panoply
of theoretical and empirical literature regarding all of the above
topics, this review focuses on one of the most diverse chemical
groups, the terpenoids, and addresses questions regarding evo-
lutionary origins, plant concentrations, and changes in func-
tion through time. Recent advances at the genetic, physiolog-
ical, and ecological scales allow us to use terpenes as models
for the study of the evolution of chemical function and of the
ecological impacts of evolutionary change. We begin by re-
viewing the broad classes of models used to explain chemical
diversity and concentration and then move on to discuss recent
advances in our understanding of terpenoids in light of these
models.
Evolutionary Models
With the recognition that secondary metabolites were not
just plant waste products came attempts to understand their
distribution and abundance. Two challenges immediately faced
efforts to develop models of allocation to secondary metab-
olites. The first was to understand the origin and maintenance
of the astounding diversity of chemicals produced in the plant
kingdom, and the second was to discover the patterns under-
lying the variation in quantities that are found both within
and among plants of the same and different species.
The diversity of chemicals found within plants has been
explained as resulting from the interaction of plants and their
major pests, pathogens, and predators. Ehrlich and Raven
(1964) proposed that the evolution of secondary plant metab-
olites and the stepwise evolutionary responses to these com-
pounds by phytophagous insects have led to the radiation and
diversity of both of these groups. While the stepwise process
of escape and radiate has come under close scrutiny, the com-
plexity of this battle between plants and insects is well doc-
umented in the diversity of secondary phytochemicals that act
as deterrents against herbivores. The model of Jones and Firn
(1991) brings advances in our understanding of chemical ac-
tivity to bear on the question of chemical diversity and indi-
cates that plants will have a much higher diversity of com-
pounds than are used for defense at any one time. Together,
the models of Jones and Firn (1991) and Ehrlich and Raven
(1964) explain the amazing chemical diversity observed. These
ecological and physiochemical models must, however, be eval-
uated within a phylogenetic framework when attempts are
made to apply them to empirical data (Monson 1996).
The more challenging issue has been, and continues to be,
developing a model for predicting the quantities of secondary
metabolites found in plants. The work of Herbert Simon to
extend optimality modeling and linear programming tech-
niques from economics to other disciplines (Simon 1959) soon
found a receptive home in ecology and evolutionary biology.
The first efforts to use optimality theory in plants were applied
to models of reproduction (Cohen 1966). Seminal articles fol-
lowed shortly thereafter that addressed questions regarding
allocation to secondary metabolites from an optimality per-
spective (McKey 1974). These articles were based on the log-
ical conclusion that any resources allocated to defense could
not be used for growth or reproduction. Thus, in evolutionary
terms, there must be a cost to defense, and this cost can be
calculated by estimating the foregone reproduction resulting
from allocation to defense. The benefit of allocation to defense
is then the increase in reproduction that occurs as a result of
a plant’s not losing biomass to pests or pathogens.
Coincident with this explicit optimality approach, Feeny
(1976) developed a model of allocation to secondary metab-
olites that was based on a notion of the risk faced by plants.
This model, the apparency model, partitions plants into those
that are likely to be specialized on by herbivores (plants that
are apparent) in contrast with those that are not. The “un-
apparent” plants, ephemeral in their distribution, are more
likely to evade detection by specialists and can afford defenses
that work at low concentrations and are mobilized for reuse.
The concept of apparency brings explicit consideration of risk
to the calculation of optimal levels of defense in plants.
In the early 1980s attention began to focus on the role of
resource availability as a parameter affecting growth rate and
the relative costs and benefits of producing secondary metab-
olites for defense. This optimality approach is seen in the evo-
lutionary model of Coley et al. (1985) and Bloom et al. (1985).
A decade ago Zangerl and Bazzaz (1992) brought together risk
and resource availability into a comprehensive optimality
model that has proven extremely effective at predicting allo-
cation to defense.
Among the models not based on optimality principles, per-
haps the most significant model was put forth in the early
 THEIS & LERDAU—EVOLUTION OF FUNCTION IN SECONDARY METABOLITES
1980s by John Bryant et al. (1983). They proposed that al-
location to various defensive compounds was governed by ba-
sic stoichiometric requirements and that when resources were
available in excess of a plant’s ability to use them for growth
or reproduction, the resources would be allocated to secondary
metabolites. This model, termed the carbon-nutrient balance
hypothesis, has excited much heat and a little light, but its
intellectual status is more akin to the sort of null model that
all good optimality models must posit simultaneously (Beatty
1980) than to a general challenge to optimality thinking (Ler-
dau and Coley 2002).
The challenge now facing ecologists and evolutionists is less
to discern which particular optimality model is most applicable
to particular plants and their secondary metabolites than it is
to bring together advances in the genetic and physiological
bases of secondary metabolite function with ecological and
phylogenetic patterns of compound distribution. For terpenes
enough is known, as we outline below, to begin to draw these
connections and develop a theoretical perspective that uses
ecology and evolution to understand diversity and abundance
from the genetic to the phylogenetic scale and that takes into
account the functional roles of these compounds.
Genetic Details and Biochemistry of Terpenoids
The functional diversity of chemicals within plants is best
demonstrated by terpenoids. More than 30,000 terpenoids
(Buckingham 1998) have been identified, each one composed
of 5-carbon (prenyl diphosphate) building blocks added
together to make 10-carbon, 15-carbon, and even 2000–
500,000-carbon chains, as is the case for rubber. Terpenes are
functionally diverse, and many are integral to primary metab-
olism, e.g., hormones such as gibberellins and abscisic acid,
electron carriers such as plastoquinone and ubiquinone,
terpene-derived compounds that form structural parts of mem-
branes such as phytosterols, and photosynthetic pigments such
as carotenoids.
There are two biochemical pathways responsible for the syn-
thesis of terpenoids. In both prenyltransferases act in the con-
densation of an isoprene unit, IDP (isopentenyl diphosphate),
with an IDP isomer, DMADP (dimethallyl diphosphate), in a
head-to-tail addition. The result, prenyl diphosphates of var-
ying lengths, is then modified through dimerization and cy-
clization by TPSs (terpene synthases) to produce a diverse array
of terpenoids with an almost equally diverse array of functions.
The head-to-tail addition of isoprenoid units was initially
identified as the biosynthetic mechanism for the synthesis of
terpenoids (Ruzicka 1953). The first pathway described, one
that is common to bacteria, fungi, mammals, and plants, is
the mevalonic acid pathway (Spurgeon and Porter 1981). In
plants there are two subcellular locations for the synthesis of
terpenes, the cytosol and the plastids. It was not until 1997
that Lichtenthaler et al. (1997) discovered that the plastid-
bound pathway was distinct biochemically. They showed that
this “new” pathway, the DOXP/MEP (deoxyxylulose phos-
phate/methylerythritol) pathway, is identical to that discovered
in bacteria a few years earlier and probably is a legacy of
prokaryotic endosymbiotic ancestors (Cvejic and Rohmer
2000). Thus, in higher plants there are two pathways for gen-
erating terpenes. The classic mevalonic acid pathway produces
S95
the precursors of sesquiterpenes (C15) and multiples thereof,
for example, the triterpenes (C30), via the cytosol; and the
plastidic DOXP/MEP produces units of C10, such as the pre-
cursors of monoterpenes (C10) and diterpenes (C20), etc.
(Lichtenthaler et al. 1997). Recent evidence indicates that there
may well be sharing of intermediates across these pathways,
a sort of biosynthetic cross talk (Jux et al. 2001).
A common ancestor has been implicated for both pro-
karyotic and eukaryotic prenyltransferases (Chen et al. 1994).
These enzymes share two highly conserved regions that contain
the aspartate-rich consensus sequence DDXX(XX)D (Mc-
Garvey and Croteau 1995). This sequence has been postulated
as the binding site for the metal ion-complexed diphosphate
group (Ashby and Edwards 1990; Lesburg et al. 1997). The
electrophilic reaction mechanism proceeds through the gen-
eration of a carbocation intermediate. This mechanism is also
used by the TPSs, which share the similar DDXXD motif
(Bohlmann et al. 1998). These electrophilic reactions in TPSs
and the manner in which the resulting carbocation is quenched
give rise to the great diversity of terpenoids functioning in so
many different metabolic processes.
Evidence for the common heritage of the plant terpene syn-
thases comes from the similar reaction mechanism, as well as
from sequence similarity, including exon and intron size and
conserved sequence motifs (Bohlmann et al. 1998; Trapp and
Croteau 2001). Phylogenetic analysis of 33 TPSs from an-
giosperms and gymnosperms established that the origin of
TPSs occurred before the splitting off of the angiosperms (Bohl-
mann et al. 1998). Synthases from angiosperms and gymno-
sperms functioning in primary metabolism are more closely
related than angiosperm synthases are to each other. However,
the specific functions of monoterpene, sesquiterpene, and di-
terpene synthases involved with secondary metabolism evolved
after the divergance of the gymnosperms and the angiosperms.
For example, the limonene synthases in gymnosperms (Abies)
and angiosperms (Mentha) are not closely related but are, in-
stead, an example of convergent evolution.
S-linalool synthase, an enzyme that produces S-linalool, an
attractive component of some floral blends, is structurally the
most distinct synthase gene identified so far. This distinctive-
ness may not be surprising as the action of this enzyme is
different from other synthases, catalyzing a simple ionization,
with water trapping the carbocation rather than the inter- or
intracarbon carbon bond formation (McGarvey and Croteau
1995; Pichersky et al. 1995). However, a common origin be-
tween plant-linalool synthase and bacterial-linalool synthase
is unlikely, since it has been hypothesized that in plants this
synthase is actually a composite gene that evolved through
a recombination event between a copalyl pyrophosphate
synthase-like gene and the limonene synthase-like gene (Cseke
et al. 1998).
In general, the synthases involved in the production of ter-
penoid secondary metabolites are far more readily adaptable
than synthases involved in the production of terpenoid primary
metabolites. While gene duplication is believed to be the pri-
mary mode for evolving a new trait from an old gene (Gottlieb
1982), the evolution of function in secondary metabolites may
not require doubling; if a gene is not critical for organismal
function, there may be continuous opportunity for new func-
tions to evolve by mutation, leading to higher diversity (Pi-
S96 INTERNATIONAL JOURNAL OF PLANT SCIENCES
chersky and Gang 2000). When such a change occurs in an
isolated population it may be tied to cladogenesis. In addition,
functional diversity can arise with very little change in synthase
structure. Closely related genes can encode enzymes synthe-
sizing dissimilar monoterpenes and sesquiterpenes. Bohlmann
et al. (1997) showed that TPSs with greater than 70%–90%
identity can catalyze distinct chemical reactions, whereas other
TPSs with no more than 30% similarity may catalyze the same
reaction. By swapping regions on two synthase genes, 5-
epi-aristolochene and vetispiradiene, Back and Chappell
(1996) were able to demonstrate that just one exon is respon-
sible for substrate specificity while another exon is responsible
for the different reaction products. Another path to diversifi-
cation involves enzymes that synthesize multiple products de-
pending on protein environment. In some cases only a single
product is formed, while in other cases numerous products are
generated by the same synthase. For example, in grand fir,
Abies grandis, the substrate farnesyl diphosphate (FPP) leads
to an extraordinary diversity of end products when acted on
by constitutive synthases such as g-humulene synthase (three
major and 52 minor sesquiterpenes) or d-selinene synthase
(three major and 34 minor sesquiterpenes). In contrast, in-
ducible synthase enzymes such as d-cadinene synthase and (E)-
a-bisabolene synthase each produce a single end-product
(Steele et al. 1998). These two single-product enzymes are in-
duced in response to herbivore damage, suggesting perhaps a
general pattern of constitutive terpene synthases leading to
multiple end-products, while those that are induced in response
to damage have fewer end-products. Clearly, further work
must be done to test the generality of this pattern.
In terms of the factors regulating the quantity of terpenes
produced, the primary regulation appears to reside in the first
step of the pathway, the conversion of prenyl diphosphate into
the structurally diverse higher terpenes (McGarvey and Cro-
teau 1995). From studies on isoprene synthase in velvet bean
(Kuzma and Fall 1993), linalool synthase in Clarkia breweri
(Pichersky et al. 1994; Dudareva et al. 1996), and limonene
synthases in mint (Gersehenzon and Croteau 1993), levels of
terpene synthase activity have been correlated with the pro-
duction of terpenoids, although levels of precursors could ul-
timately be a rate-limiting step (McGarvey and Croteau 1995).
For example, the level of monoterpenes in the oil glands of
mint species reflects the enzyme levels (Gersehenzon and Cro-
teau 1993; Gershenzon et al. 2000). Similarly, linalool synthase
activity closely matches the production of linalool and its de-
rivatives. However, when petunia was transformed with S-
linalool synthase cDNA linalool was successfully expressed but
not emitted. Instead it was converted to the nonvolatile -linalyl-
b-D-glucopyranoside (Lücker et al. 2001). Induction by
wounding or by fungal elicitors may also lead to an up-
regulation of TPSs and prenyltransferases (Martin et al. 2002).
However, with the notable exception of Mentha, little is known
about the enzymes along the pathways. Without information
on concentration of intermediates and flux rates through the
pathway, it is difficult to accurately assess the most important
regulatory steps (Croteau and Gershenzon 1994).
Genetic variation (both within and among species) occurs
in the context of both epistatic interactions and simple Men-
delian relationships. For example, epistatic interactions have
been implicated for the two best-studied systems among the
angiosperms, Thymus and Mentha. In Thymus vulgaris there
are six chemotypes in which epistatic interactions form a dom-
inance hierarchy. The chemotypes, in rank order of dominance,
are geraniol 1 alpha terpineol 1 thuyanol-4 1 linalool 1 car-
vacrol 1 thymol. A series of six loci each with a dominant and
a recessive allele controls the branching along the pathway
and, thereby, the chemotype produced (Vernet et al. 1986).
Unless the plant is homozygous recessive for geraniol, geraniol
will be produced; if it is homozygous recessive for geraniol,
then alpha terpineol will be produced; if it homozygous re-
cessive for both geraniol and alpha terpineol, then thuyanol-
4 will be produced, and so on, along the hierarchy of the six
morphs.
In Mentha different species have been extensively studied to
determine the genetic control of the monoterpenes in this ge-
nus, with attention paid both to the end-products and to the
enzymes that produce the intermediates (Murray and Lincoln
1970; Hefendehl and Murray 1976; Croteau and Gershenzon
1994). Most of the genes in the pathway appear to be diallelic
with simple dominant and recessive alleles. For example, lim-
onene synthase is controlled by two diallelic genes that interact
epistatically. The interactions between these two genes control
the production of the three possible end-products: (-)-trans
carveol, (-)-trans-isopiperitenol, or limonene itself. Compli-
cating the picture further, a third allele epistatically affects these
two genes and can lead to the synthesis of linalool and linalyl
acetate rather then any of these three cyclic monoterpenes. The
study of Mentha has also led to the identification of several
multiallelic loci as well as alleles with incomplete dominance
(Croteau and Gershenzon 1994).
In addition to the complex examples outlined above, there
are a number of examples of simple genetic control by one
major dominant gene or by closely linked loci for angiosperms
as well as gymnosperms. In Perilla frutescens, there are two
dominant genes essential for the formation of cyclic monoter-
penoids (Nishizawa et al. 1992), and in Achillea simple dom-
inant genetic control has been identified for the sesquiterpene
longipinenone (Fischer et al. 2001). In Pinus elliotii, both b-
pinene and myrcene are each controlled by single but inde-
pendent genes (Squillace 1971). Similarly, d-3-carene, a mono-
terpene present in Pinus pinaster, is controlled by one major
gene or closely linked loci (Plomion et al. 1996). Experimental
studies using hybrids have produced similar results. Raguso
and Pichersky (1999) found that S-linalool in C. breweri was
controlled by simple Mendelian dominance. Shepherd et al.
(1999) used QTL analysis on half sibs and also found the
monoterpenes of a eucalypt hybrid to be controlled by a single
gene region. In summary, it is difficult to generalize the relative
importance of simple Mendelian compared with complex ep-
istatic relationships in determining the composition and quan-
tities of terpenes produced. The apparent ubiquity of both
mechanisms, however, indicates multiple possibilities for ge-
netic control, and, thus, evolutionary change, in most taxa.
Location and Function of Terpenoid Production
Terpenoid secondary metabolites occur across a wide range
of plant tissue types; those serving as defensive chemicals are
often secured in secretory structures. Such specialized struc-
tures minimize the risk of autotoxicity but maintain terpene
 THEIS & LERDAU—EVOLUTION OF FUNCTION IN SECONDARY METABOLITES
concentrations at sites whose defense is crucial. In conifers,
terpenoids are stored in resin ducts or lactifers under pressure,
which can have a devastating impact on small herbivores (Ger-
shenzon and Croteau 1991). In angiosperms, glandular tri-
chomes that contain terpenes often serve as a first line of de-
fense against would-be herbivores. These externally based
structures enhance the effectiveness of chemical defenses be-
cause they can arrest feeding preemptively. In addition, pellucid
glands and secretory ducts within leaves are also common
(Fahn 1988).
In conifers, oleoresin, a complex mixture of terpenoids that
includes monoterpenes, sesquiterpenes, and diterpenes, is an
important defense strategy against bark beetles and their as-
sociated fungal pathogens (Langenheim 1994). There is great
diversity in the manner of storage and release for this defensive
blend. On one end of the continuum are compounds stored in
branched interconnected canals, e.g., the resin canal system of
Pinus. Attack by an herbivore or pathogen causes very little
biochemical induction of terpene synthesis in conifers with
complex canal systems; the main plant response involves the
transport of terpenes through the canals to the site of attack,
often across several meters. At the other end of the spectrum
are the simple resin cells of Thuja (cedar), which can be induced
by attack but are normally either absent or present in very low
quantities. Abies (true firs) have multicellular resin blisters that
are less complex in Larix, Picea, and Pseudotsuga (larch,
spruce, and Douglas fir), where the blisters are connected but
also inducible (Lewinsohn et al. 1991).
As Gershenzon and Croteau (1991) point out, the defense
theory proposed by McKey (1979) and Rhoades (1979) of
“risk and value” has largely proven useful in locating the tis-
sues with the highest levels of terpenes. Concentrations of ter-
penoids are generally higher in reproductive structures and
typically highest in the foliage during and immediately follow-
ing flowering (Rapparini et al. 2001). Young leaves are more
important to a plant than older leaves, and the highest levels
of terpenoids are found in the young organs (Fischbach et al.
2002). Variation in concentration can also vary within a tissue.
The pattern of constitutive monoterpenes along the needles of
ponderosa pine slows caterpillar feeding. Terpenes increase as
the caterpillar feeds, motivating the caterpillar to switch nee-
dles and slowing the rate of damage, thereby giving the plant
time to induce TPSs (Litvak and Monson 1998).
For terpenoids that are released to attract insects, specialized
storage structures are far less common. These compounds are
generally released as they are produced and so are maintained
within plant tissues far below toxic levels. However, there are
exceptions to this pattern. Osmophores are specialized scent-
producing organs of variable morphology and ontogeny, many
of which store the volatiles they produce. In orchids such as
Stanhopea, Clowesia, and Polycycnis, euglossine bees are at-
tracted to the terpene-based scent that is emitted from these
structures (Vogel 1963; Dressler 1982). Specialized structures
are not necessary for the establishment of spatial patterns in
floral emissions. Pollen and anthers can have altogether dif-
ferent mixtures of volatile scents compared to the rest of the
flower, and many pollen-feeding insects are adapted to dis-
cerning these scent mixtures (Dobson 1987; Dobson and
Bergstrom 2000). Scent guides that bolster visual nectar guides
have also been identified, indicating that interactions among
S97
sensory modalities may be crucial in determining the function
of particular terpenes (Bergstrom et al. 1995).
Dalechampia is an excellent example of the crucial role that
spatial patterns of scent emission can play in plant-insect in-
teractions. From a phylogenetic study, Armbruster et al. (1997)
determined that a triterpene resin underwent an evolutionary
switch in function from a deterrent and toxin to an attractant
and reward, and then back again to its deterrent role. Ances-
trally, resin functioned as a defense, evenly distributed in the
floral tissue. However, in a group of more derived species
within the same genus, pollinated by resin-collecting bees, resin
ducts are unified and positioned for optimal pollinator at-
traction leading to greater pollination efficiency. In the most
derived species, resin ducts occur in the leaves and protect the
foliage from herbivores. The chemical composition of the res-
ins is similar in all three taxa; it is the placement of the resin
duct within the plant that defines its functions.
Organism Response and the Evolution of
Function in Terpenoids
For all compounds involved with interorganismal signaling,
function can evolve through changes in the recipient organisms
rather than through changes in the sending organism. The
literature is replete with accounts of both toxicity and repel-
lency on insect, mammalian, and molluscan herbivores (Ger-
shenzon and Croteau 1991; Harborne 1993; Langenheim
1994). These toxic effects have been demonstrated mostly for
generalist herbivores, but many of these studies have been con-
ducted on agricultural pests rather than actual herbivores the
plant is likely to encounter in a natural setting (Berenbaum
1986; Gershenzon and Croteau 1991). Many specialist insects,
which the plant certainly does encounter, have seemingly
adapted to these defensive compounds. No longer do they find
these compounds repellent; instead the compounds may be
used as cues for host recognition, even when they retain their
growth inhibiting effects. While feeding on the toxin may re-
duce growth, fitness can actually increase through reduced pre-
dation due to sequestered toxins.
Are these adaptations and counteradaptations the very phe-
nomena that Ehrlich and Raven envisioned in 1964? Few tax-
onomic groups have been put to the test, because a sound test
requires both a robust phylogeny and ample ecological infor-
mation. Most of the research conducted on plant-insect inter-
actions has not explicitly addressed even reciprocal adaptation
(Futuyma 2000), much less the “escape and radiate” hypoth-
esis of Erhlich and Raven. In the few cases where it has been
properly tested, the hypothesis has been both rejected, e.g.,
Mexican flea beetles, Blepharida, on its host Bursera (Becerra
1997), and supported, e.g., Phyllobrotica beetles and plants
in the Lamiales (Farrell and Mitter 1990) and Tetraopes (long-
horned beetle) on Asclepias (milkweed) (Farrell and Mitter
1998). This last example appears to follow a phylogenetic
progression in the potency of the cardenolides, tracked by the
counteradaptations of the beetle lineage. These linked changes
are especially intriguing as the beetles are just one of the many
groups of herbivores that feed on Asclepias.
The most charismatic herbivore on Asclepias is certainly the
monarch butterfly Danaus plexippus, which is the textbook
example of a specialist herbivore that feeds on a toxin (car-
S98 INTERNATIONAL JOURNAL OF PLANT SCIENCES
denolides) and gains advantage via sequestration. Interestingly,
for first instar larvae, a high concentration of cardenolides
actually reduced the rate of growth and survival (Zalucki and
Malcolm 1999). Another specialist on Asclepias, the weevil
Rhyssomatus lineaticollis, is cryptically colored and does not
sequester the toxin, although there are high concentrations of
cardenolides in the pith where the larvae feed. The stems on
which adults lay their eggs have relatively lower concentrations
(Fordyce and Malcolm 2000). These two alternative tactics
highlight the flexibility that exists when natural selection can
act on two operators; the strategy of feeding on Asclepias leads
to very different ecological tactics that, in turn, are correlated
with extreme differences in morphology, physiology, and
behavior.
A number of herbivores recycle terpenoids, using them for
protection without sequestering them; they secrete the ter-
penes, and this secretion is used to ward off predators. Some
species of the plant genus Bursera produce a resin after insect
damage; specialist herbivores in the genus Blepharida utilize
this resin by adorning their backs with a regurgitant and an
anal secretion that match the hosts’ defense chemistry (Evans
et al. 2000). The weevil Oxyops vitiosa produces a mixed
terpenoid secretion garnered from its host plant Melaleuca
quinquenervia (Wheeler et al. 2002). By covering its integu-
ment with this secretion, the weevil repels generalist predators.
Another beetle, Eurypedus nigrosignata, constructs fecal
shields using terpenes found in their larval host plant Cordia
curassavica (Gomez et al. 1999). Similarly, larvae of the leaf-
feeding beetle Lema trilinea carry fecal shields that contain
steroidal glycoalkaloids and saponins, which are modified
from their host Solanum dulcamara (Morton and Vencl 1998).
For volatile compounds such as the monoterpenes and ses-
quiterpenes, it is likely that the concentration of the compound
is related to its function. As noted above, terpenoids whose
function is involved with reducing herbivory tend to be stored
in secretory structures or organs, which usually contain ter-
penes at high concentrations, on the order of milligrams per
gram (Kleinhentz et al. 1999). Terpenoids involved with pol-
linator attraction, however, tend to be located in parts of the
plant where the resistance to flux is very low, and substantial
flux rates can occur even at low concentrations, on the order
of micrograms per gram (Schiestl and Ayasse 2000).
Changes in function through changes in receiver underlie
some of the most important events in the evolution of land
plants. During the Carboniferous period, ca. 300 Ma, flower-
like structures and insects capable of pollination began to
emerge. By the Cretaceous, 150 million years later, pollen and
ovules supplemented the diets of some predatory insects (Cre-
pet 1983). The chemical deterrents against pests and pathogens
emitted by plants were co-opted by insects seeking either food
or mates (Pellmyr and Thien 1986). The chemistry of floral
scents is similar to nonfloral deterrents, and theory indicates
that these early herbivorous insects inadvertently acted as pol-
linators; thus a novel selection pressure arose for chemical
volatiles to be attractive (Pellmyr and Thien 1986). In a study
of the scent emitted by insect-pollinated cycads, representing
an independent evolution of insect attractants, Pellmyr et al.
(1991) found classes of attractant compounds that are chem-
ically similar to those produced by taxa with deterrent
volatiles.
Phylogenetic analyses of floral scent have demonstrated that,
while there is some conservation in the classes of compounds
produced, there is also a high degree of homoplasy at the level
of the specific compound (Barkman 1997; Levin et al. 2001).
The great diversity of compounds is likely maintained to pro-
mote pollinator fidelity. When rewards are not forthcoming,
the importance of these signals is especially apparent and pol-
linator attraction occurs by deceit. For example, in Ophrys
spp. (Orchidaceae) chemical mimics of specific insect phero-
mones are emitted by the orchids in order to attract male
pollinators (Ayasse et al. 2000; Mant et al. 2002). Some Aroids
attract pollinators seeking oviposition sites by emitting an odor
of carrion or excrement (Kite 1995; Kite and Hettersheid
1997). Specificity of floral scent can be beneficial by encour-
aging fidelity, as in the extreme case of the figs and their fig
wasp pollinators. It is likely that each of the 750 species of fig
emits a blend specific enough to attract its one species of fig
wasp (Hossaert-McKey et al. 1994; Grison-Pige et al. 2002).
In geonomoid palms, there is also evidence for maintenance
of reproductive isolation through pollinator attraction (Knud-
sen 1999), and such relationships have been shown for a va-
riety of taxa, including but not limited to Ranunculaceae, Cim-
icifuga (Pellmyr 1986) and Orchidaceae, Satyrium (Ellis and
Johnson 1999).
In what appears to be the most recently derived function of
volatile terpenes, the emission of typical “floral” compounds
is induced from the foliage of cotton, tobacco, and corn by
the feeding of larval lepidoterans. Parasitoid wasps, in turn,
utilize this signal to locate their larval lepidopteran hosts. Upon
positive identification, the wasp will oviposit into the feeding
caterpillar. The specificity of this signal has been demonstrated;
Heliothis virescens and Helicoverpa zea feeding on corn each
elicit the production of a unique ratio/blend of terpenoids, as
well as lipoxygenase-derived volatile compounds. The differ-
ence in the two signals is distinguished by the specialist par-
asitoid Cardiochiles nigriceps and allows it to pinpoint its host,
H. virescens (De Moraes et al. 1998). Similarly, the parasitoid
Cotesia glomerata uses the signals produced by Brassica oler-
aceae var. gemmifera (brussel sprouts), where the emission of
green leaf volatiles as well as sesquiterpenes and cyclic mono-
terpenoids is systemically induced after feeding by Pieris bras-
sicae caterpillars (Mattiacci et al. 2001).
One of the most exciting recent discoveries involving volatile
terpenes and signaling to predators and parasitoids involves
the apparently ubiquitous induction of homoterpenes in re-
sponse to herbivore damage (Degenhardt and Gershenzon
2000). Homoterpenes are C-11 and C-16 compounds pro-
duced by the removal of four carbons from sesquiterpenes and
diterpenes. In certain plants, predators and parasitoids have
learned to use the induced release of these compounds as cues
to locate prey/hosts. It appears that this induction may be
primitive across higher plants (Boland et al. 1998) but that
the insect response may have been independently derived mul-
tiple times. Further study on concerted phylogenies of plants,
herbivores, and their predators/parasitoids is necessary to de-
termine whether or not these these relationships involve mul-
tiple independent evolutions of a novel function.
This “cry for help” has been demonstrated for a number of
different plants soliciting predators. Linalool is induced in
Phaseolus lunatus, lima bean, following infestation by the her-
 THEIS & LERDAU—EVOLUTION OF FUNCTION IN SECONDARY METABOLITES
bivorous mites Tetranychus urticae. The herbivore is repelled
by this compound but not so the predatory mite, Phytoseiulus
persimilis (Dicke 1986). In another example, sesquiterpenes
are induced by potato plants that have been damaged by the
Colorado potato beetle, Leptinotarsa decemlineata, and Weiss-
becker et al. (2000) found the predaceous stink bug, Perillus
bioculatus, which is attracted to damaged plants, to be re-
sponsive to these compounds. Predator cuing in on insect dam-
age has also been demonstrated for conifers. Oxygenated
monoterpenes attract the bark beetle parasitoid Roptrocerus
xylophagorum after insect damage by two herbivores, the
southern pine beetles and the southern pine engraver, in Pinus
taeda, loblolly pine (Petterson et al. 2000). Similarly, in spruce
the bark beetle parasitoid Rhopalicus tutela is attracted to bark
beetle larvae “concealed” under the bark of spruce trees by
the emitted volatiles (Pettersson 2001).
The induction of volatile compounds as a result of insect
feeding sets up the possibility that neighboring plants could,
in fact, use these signals, much as parasitoids do, to detect
insect presence on neighboring plants. This intriguing idea de-
scribed as “talking trees” has been met with much controversy
since it was first introduced 20 years ago (Baldwin and Schultz
1983; Rhoades 1983). Proper controls have been lacking in
many studies that find plants responding to neighboring in-
duction (Dicke and Bruin 2001). Recently, Karban et al. (2000)
demonstrated in manipulated field conditions that when sage-
brush, Artemesia tridentate, emits an epimer of methyl jas-
monate after mechanical damage, neighboring Nicotiana at-
tenuata individuals respond by inducing defense compounds.
The methyl jasmonate produced by the damaged plants has
been shown to increase resistance to herbivores (Farmer and
Ryan 1990). The neighboring wild tobacco plants, which grow
in close proximity naturally, increased their synthesis of
polyphenol oxidase, an enzyme putatively involved with de-
fense, and these plants experienced less herbivory than control
plants. This response disappeared when the air between plants
was blocked but not when the soil was blocked. Recently, in
a lab-based study lima bean has demonstrated plant-to-plant
communication through a volatile signal. In the presence of
an herbivore-damaged plant, the genes of undamaged lima
bean elevate the expression of several terpenoids (Arimura et
al. 2000). Arguments from physical and chemical transport
principles, however, suggest that plant-to-plant communica-
tion by volatile signals is probably exceedingly uncommon
(Lerdau 2002).
Conclusion
It is clear even from this brief review of terpenoids that the
evolution of function in secondary metabolites is both a com-
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plex and potentially rewarding field of study. The complexity
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Acknowledgments
We received support during the preparation of this manu-
script from the National Science Foundation (DEB 0206300),
the Theodore Roosevelt Memorial Fund of the American Mu-
seum of Natural History, the Bullard Fellowship and the Hrdy
Conservation Biology Programs of Harvard University, and
the Andrew Mellon Foundation. We thank Diane Martin, Rob
Raguso, and two anonymous reviewers for comments on an
earlier draft.
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