Coevolution of generalist feeding ecologies and
gyrencephalic mushroom bodies in insects
Sarah M. Farris*† and Nathan S. Roberts‡
*Department of Biology, West Virginia University, P.O. Box 6057, Morgantown, WV 26506; and ‡Program in Biochemistry, Washington and Jefferson
College, 51 Lincoln Hall, Washington, PA 15301
Communicated by Gene E. Robinson, University of Illinois at Urbana–Champaign, Urbana, IL, September 27, 2005 (received for review June 22, 2005)
Here we demonstrate the independent acquisition of strikingly
similar brain architectures across divergent insect taxa and even
across phyla under similar adaptive pressures. Convoluted cortical
gyri-like structures characterize the mushroom body calyces in the
brains of certain species of insects; we have investigated in detail
the cellular and ecological correlates of this morphology in the
Scarabaeidae (scarab beetles). ‘‘Gyrencephalic’’ mushroom bodies
with increased surface area and volume of calycal synaptic neuropils and increased intrinsic neuron number characterize only those
species belonging to generalist plant-feeding subfamilies, whereas
significantly smaller ‘‘lissencephalic’’ mushroom bodies are found
in more specialist dung-feeding scarab beetles. Such changes are
not unique to scarabs or herbivores, because the mushroom bodies
of predatory beetles display similar morphological disparities in
generalists vs. specialists. We also show that gyrencephalic mushroom bodies in generalist scarabs are not associated with an
increase in the size of their primary input neuropil, the antennal
lobe, or in the number of antennal lobe glomeruli but rather with
an apparent increase in the density of calycal microglomeruli and
the acquisition of calycal subpartitions. These differences suggest
changes in calyx circuitry facilitating the increased demands on
processing capability and flexibility imposed by the evolution of a
generalist feeding ecology.
afferent ! behavior ! carnivore ! sociality
T
he insect mushroom bodies, paired neuropils that act as
centers for sensory discrimination, integration, and associative and flexible behaviors, are easily recognized due to their
characteristic cellular architecture (1–5). Mushroom bodies are
composed of thousands of tightly packed intrinsic neurons called
Kenyon cells. Kenyon cell dendrites are grouped into dorsally
positioned neuropils termed calyces, which serve as dedicated
afferent input regions and receive a preponderance of olfactory
afferents from the antennal lobe and, in some taxa, visual and"or
gustatory afferents as well (6–8). Kenyon cell axons project
ventro-anteriorly to the pedunculus and typically bifurcate into
combined input"output structures, the medial and vertical lobes.
Although all insect mushroom bodies share a basic groundplan, substantial morphological and presumably functional modifications are readily observed (9). Morphological variability of
mushroom bodies often reflects species-specific behavioral ecology. In the social Hymenoptera (including the ants, bees, and
wasps), the calyx is partitioned into afferent zones characterized
by the dendrites of Kenyon cell subpopulations and by sensory
input modality (6, 10, 11). Two of these calyx zones, the lip and
the collar, receive olfactory and visual input, respectively. In
ants, the relative size of the lip and collar vary with the size of
their primary sensory input neuropil, the antennal and optic
lobes, and also with the importance of each sensory modality in
the life of the animal (12). Similar correlations between the
periphery and higher processing centers have been observed in
mammals. In the star-nosed mole, deemphasis of vision as a
primary sensory modality has led to reduction of the eyes and
visual cortex, whereas somatosensory cortex has greatly ex17394 –17399 ! PNAS ! November 29, 2005 ! vol. 102 ! no. 48
panded to accommodate the processing needs of highly specialized somatosensory processing structures on the snout (13).
Distribution of anatomical features across more phylogenetically diverse insect taxa suggests independent origins, perhaps in
response to similar adaptive pressures, in these lineages. This is
dramatically illustrated by the distribution of single and double
primary calyx neuropils. Single ovoid calyces are found in the
firebrats, representative of the most basal extant insect order,
and the flies, one of the most derived groups (14–17). Doubled,
deeply cup-shaped calyces are observed in widely divergent
groups, such as the hemimetabolous cockroaches and the holometabolous social Hymenoptera (9).
What are some potential causal mechanisms and adaptive
significances of calyx doubling? At the cellular level, mushroom
bodies of cockroaches and social Hymenoptera are the largest
known, with !170,000–175,000 Kenyon cells per hemisphere
(18, 19). Calyx volume is likely to be large relative to the rest of
the brain in these insects, compared with those possessing fewer
Kenyon cells. Spreading of the calyx into two separate neuropils
and convolution of the synaptic neuropil may be a consequence
of maintaining optimal surface area to volume ratios in large
mushroom bodies. Such a mechanism has been proposed for the
formation of cortical gyri and sulci (gyrification) in mammals,
which is pronounced in groups with relatively large cortices such
as primates and cetaceans (20, 21). Developmental studies
provide additional support for a positive correlation among
cortex volume, neuron number, and gyrencephaly in mammals.
For example, decreased neuronal number is observed in human
patients and mouse mutants with lissencephalic (smooth cortex)
mutations (22), whereas increasing the number of neural precursors can generate an abnormally large number of gyri and
sulci in the cortex of the mouse (23).
We tested the hypothesis that double mushroom body calyces,
like cortical gyri and sulci, are associated with an increase in
mushroom body size relative to single calyces in terms of Kenyon
cell number, calyx neuropil surface area and calyx volume. We
accomplished this by quantifying mushroom body architecture in
scarab beetles (Coleoptera: Scarabaeidae), species of which may
possess either single or double calyces (24), as illustrated in Fig.
1 A and B. We also asked whether calyx morphology in scarab
beetles was associated with changes in its most prominent source
of synaptic input, the antennal lobes (25). Finally, we mapped
calyx structure according to phylogeny and feeding ecology in
other Coleoptera to determine potential behavioral correlates of
calyx diversity in insects.
Materials and Methods
Insects and Histology. A total of 48 individual beetles representing
six scarabaeid subfamilies and 11 different species (see Table 1,
which is published as supporting information on the PNAS web
site, for summary information on species identity, feeding ecolConflict of interest statement: No conflicts declared.
†To
whom correspondence should be addressed. E-mail: [email protected].
© 2005 by The National Academy of Sciences of the USA
www.pnas.org"cgi"doi"10.1073"pnas.0508430102
ogy, calyx morphology, and sample sizes) were collected as adults
in the Morgantown, WV, area or ordered from Hatari Invertebrates (Portal, AZ). The nonscarabaeids Geotrupes sp.
(Geotrupidae), Trox sp. (Trogidae), Odontotaenius disjunctus
(Passalidae), Sphaeridium sp. (Hydrophilidae) and Silpha sp.
(Silphidae), Scarites subterraneus (Carabidae), Scaphinotus elevatus (Carabidae), and Harmonia axyridis (Coccinellidae) were
also collected in or around Morgantown or obtained from
Carolina Biological Supply Company (Burlington, NC). Insects
were anesthetized with cold and brains dissected in physiological
saline, followed by fixation in Carnoy’s fixative at 20° C. Fixation
was allowed to proceed for a duration ranging from 1 h 15 min
to 1 h 45 min to keep tissue shrinkage between brains relatively
constant. After fixation, brains were stored in 70% ethanol at 4°C
overnight. The next day, dehydration through a graded series of
ethanols (10 min each) was followed by clearing in xylene (2 "
10 min) and paraffin embedding in a frontal orientation. EmFarris and Roberts
bedded brains were sectioned at 10 !m on a rotary microtome
and stained by using Cason’s stain according to the protocol of
Kiernan (26) to reveal brain architecture. Brains of both male
and female beetles were analyzed, with the exception of antennal
lobe glomerulus counts in which only male brains were used. The
age of beetles sampled was not known, but adult mushroom body
neurogenesis appears to be negligible or absent for these species
(S.M.F. and J. Miller, unpublished data) and is thus unlikely to
be a source of significant variation.
Volume and Cell Number Estimates. Measurements were made in one
randomly selected hemisphere of each stained brain. Brain region
volumes were calculated according to the Cavalieri method (27–29),
which has been demonstrated to provide accurate estimations of
volume from sectioned material by taking into account sampling
frequency and section thickness. First, areal measurements of brain
regions were directly measured from photomicrographs by using
PNAS ! November 29, 2005 ! vol. 102 ! no. 48 ! 17395
EVOLUTION
Fig. 1. Single and double calyces of scarabaeid mushroom bodies. (A) Single calyx of Onthophagus hecate (Scarabaeinae). (B) Double calyx of Maladera castanea
(Melolonthinae) (Scale bars: 20 !m.) (C–F) Average brain measurement ratios for beetles with single calyces (black bars) vs. double calyces (white bars). (C) Calyx volume
relative to central brain volume. (D) Kenyon cell number relative to central brain volume. (E) Measured calyx surface area relative to predicted calyx surface area. (F)
Calyx surface area relative to calyx volume. Brackets, calyces; white arrowheads, sensory input to the calyx from the antennal lobe; black arrowheads, calycal
subcompartments. *, Significant difference (P " 0.05) as determined by one-way ANOVA. Error bars in C–F represent standard errors of the mean.
Fig. 2. Distribution of calyx morphologies and feeding ecologies across the Coleoptera. The phylogenetic tree of the Scarabaeoidea is after ref. 30; the
remainder of the coleopteran tree is after ref. 49. The family Scarabaeidae is considered a monophyletic grouping. (Scale bars, located at the bottom right of
each photomicrograph, represent 20 !m for brains of Silphidae, Hydrophilidae, Trogidae, and Geotrupidae and 50 !m for Passalidae and Scarabaeidae.)
Zeiss AXIOVISION 4 software (Zeiss, Oberkochen, Germany). Sampling frequency for these measurements was determined for each
brain region [Kenyon cell body region, mushroom body calyx
neuropil (excluding the pedunculus), antennal lobe neuropil, and
total central brain] for each species to provide a volume estimate
within 5% of that generated by measurements by using every 10-!m
section. Typical sampling frequencies ranged from every second
section (calyx neuropil surface areas and volumes in the smallest
species) to every sixth section (total central brain volume in the
largest species). Once sampling frequency was determined, the first
section to be sampled in each brain was chosen by tossing a number
of coins equal to the sampling frequency; the number of ‘‘heads’’
was the first section to be sampled after the appearance of the
structure of interest.
Perimeter measurements of the calyx neuropil and diameters
of individual Kenyon cell bodies were also measured from
photomicrographs by using the Zeiss AXIOVISION 4 software.
Surface area of the synaptic neuropil of the calyx was calculated
from perimeter measurements multiplied by section thickness.
Sampling frequency for perimeter measurements was determined in the same manner as for volume estimations.
To calculate Kenyon cell number, 50–100 individual Kenyon
cell soma were selected from throughout the Kenyon cell body
region and their diameters measured by using the AXIOVISION 4
software. An average Kenyon cell body diameter was calculated
from these data and subsequently used to calculate an average
Kenyon cell body volume for that brain. The total volume of the
Kenyon cell body region was then divided by the average
individual Kenyon cell body volume to provide an estimate of
Kenyon cell number for that brain.
Finally, the total number of antennal lobe glomeruli was
counted from serial sections of male brains only, to control for
the likely presence of sex-specific glomeruli.
Measurement calculations and graphs were generated by using
Microsoft EXCEL X for Mac (Microsoft, Redmond, WA), then
exported to JMP software (SAS Institute, Cary, NC) for statistical
analyses. Measured calyx surface areas were compared with
calculated calyx surface areas, the latter derived from the
measured calyx volume and the equation for the surface area of
a sphere (# 4#r2). Brain measurement ratios for species with
single vs. double calyces were tested for significant differences by
using one-way ANOVA. All statistical comparisons were considered significant at P " 0.05.
17396 ! www.pnas.org"cgi"doi"10.1073"pnas.0508430102
Results
Double (‘‘gyrencephalic’’) calyces in scarab beetles were positively associated with a significant increase in calyx volume
relative to total central brain size when compared with single
(‘‘lissencephalic’’) calyces (Fig. 1C; one-way ANOVA F #
124.999, P $ 0.0001). Kenyon cell number was also dramatically
increased in gyrencephalic mushroom bodies (Fig. 1D; one-way
ANOVA F # 79.520, P $ 0.0001). The measured surface areas
of lissencephalic calyces were nearly identical to those predicted
for spheres of the same volume, resulting in a measured to
predicted surface area ratio of !1 (black bar, Fig. 1E). In
contrast, the ratio of measured calyx surface area to predicted
calyx surface area was significantly larger for gyrencephalic
calyces (white bar, Fig. 1E; one-way ANOVA F # 46.684, P $
0.0001). Surface area to volume ratios, however, were significantly higher in lissencephalic calyces (Fig. 1F; one-way ANOVA
F # 51.556; P $ 0.0001). This suggests that, although gyrencephaly increases the surface area of the calyx neuropil, it has not
kept pace with the expansion in calyx volume in scarabaeids.
We discovered that calyx morphology was tightly linked to
feeding ecology; lissencephalic calyces were always observed in
scarabaeid species belonging to dung-feeding (coprophagous) subfamilies, whereas gyrencephalic calyces were always observed in
plant-feeding (phytophagous) subfamilies (Fig. 2; also see supporting information) (30). Phytophagous scarabs are highly generalist,
feeding on the foliage, flowers, and fruits of a wide range of host
plants. By contrast, coprophagous scarabs and species belonging to
related nonphytophagous taxa are relatively more specialized in
their feeding habits and exhibited lissencephalic calyces. The woodfeeding Passalidae, however, were exceptional in possessing somewhat enlarged and flattened calyces, perhaps reminiscent of an
early stage in calyx splitting. Nevertheless, the overall distribution of
calyx morphologies across the taxa sampled strongly supports the
inference that robustly gyrencephalic calyces evolved in accompaniment with a derived and highly generalist feeding ecology in the
Scarabaeidae.
Additional comparisons of scarab calyces and their major
sensory input source, the antennal lobes, provided further insight
into the potential behavioral significance of large mushroom
bodies in generalist scarabs. We found that antennal lobe volume
relative to total central brain volume was actually larger in
beetles with lissencephalic calyces than in those with gyrencephalic calyces (Fig. 3A; one-way ANOVA F # 12.306, P # 0.001),
and there was no difference in the number of antennal lobe
Farris and Roberts
glomeruli in males (Fig. 3B; one-way ANOVA F # 0.048, P #
0.829). This suggests that calyx gyrencephaly is unlikely to be
associated with greatly increased numbers of antennal lobe
neurons projecting to the calyx.
Instead, our data suggested that individual afferent neurons
might form synapses with more Kenyon cells in the mushroom
bodies of generalist species. Generalist scarabs had nearly four
times as much calyx volume per unit of antennal lobe volume
compared with coprophagous scarabs (Fig. 3C; one-way
ANOVA F # 133.017, P $ 0.0001) and significantly more
Kenyon cells per unit of calyx volume (Fig. 3D; one-way ANOVA
F # 4.883, P # 0.032). This latter measurement was supported
by high-magnification images of calycal microglomeruli, sites of
Farris and Roberts
convergence of Kenyon cell dendrites, and extrinsic inhibitory
processes about a single antennal projection neuron terminal
(31). Microglomeruli were smaller and more densely packed in
the calyces of generalist scarabs (Fig. 3 E and F). Calycal
subcompartments were also observed in gyrencephalic mushroom bodies (Fig. 3E), perhaps indicating the segregation of
olfactory processing inputs or acquisition of other afferent
modalities such as vision or gustation.
Discussion
Our findings suggest that gyrencephalic calyces in scarabaeid
beetles, and perhaps other lineages such as the cockroaches and
the social Hymenoptera, have arisen in conjunction with an
PNAS ! November 29, 2005 ! vol. 102 ! no. 48 ! 17397
EVOLUTION
Fig. 3. Differences in calycal circuitry in single and double calyces. (A–D) Average brain measurement ratios for beetles with single calyces (black bars) versus
double calyces (white bars). (A) Antennal lobe volume relative to central brain volume. (B) Number of antennal lobe glomeruli (male beetles only). (C) Calyx
volume relative to antennal lobe volume. (D) Calyx volume relative to Kenyon cell number. Error bars in A–D represent standard errors of the mean. *, Significant
difference (P " 0.05) as determined by one-way ANOVA. ns, no significant difference. (E) Microglomeruli in the double calyx of Popillia japonica (Rutelinae).
Two sizes of microglomeruli (small and large circles) occupy distinct calycal subcompartments (separated by black line). (F) Microglomeruli (circles) in the single
calyx of Canthon sp. (Scarabaeinae). (Scale bars: 10 !m.)
Fig. 4. Differences in calyx structure in generalist and specialist predators. (A) Tripartite gyrencephalic calyx (brackets) of the generalist predator Scarites
subterraneus (Carabidae). (B) Lissencephalic calyx of the carabid Scaphinotus elevatus, a specialist predator of snails and slugs. (C) Minute lissencephalic calyx
of the ladybird beetle Harmonia axyridis (Coccinellidae), a specialist predator of aphids and scale insects. Arrowheads, Kenyon cell body regions. (Scale bars: A,
50 !m; B and C, 20 !m.)
evolutionary increase in Kenyon cell number, neuropil volume,
and neuropil surface area. Similar trends are observed in
mammals and are particularly pronounced in lineages such as
primates, in which increased size of the cerebral cortex relative
to total brain size is strongly correlated with an increase in gyral
complexity (20, 21). Nonhomologous brain centers like the
mushroom bodies and the cerebral cortex can thus adopt strikingly similar architectures when shared principles of cellular
organization are acted on by similar selective pressures.
Gyrencephalic calyces are not associated with a concomitant
increase in the volume of the antennal lobes, suggesting that the
evolution of large mushroom bodies in highly generalist phytophagous beetles was not driven by the acquisition of inputs from
more olfactory afferent neurons. Instead, gyrencephalic calyces
may represent an adaptation for enhancing the computational
capacity of the mushroom bodies in three ways: by increasing the
number of inputs from individual afferent neurons, by subcompartmentalization for segregation of specialized functions, and
by providing additional substrate for complex learning computations and the storage of memories. Regarding the first possibility, modeling studies have proposed that small groups of
antennal projection neurons converge combinatorially on populations of Kenyon cells to form odor-discriminating ‘‘functional
subunits’’ (32). Increasing the number of Kenyon cells would
thus generate a greater capacity for combinatorial synaptic
inputs from the antennal lobe and the potential to discriminate
among larger numbers of odors. Regarding the second possibility, calycal subcompartments like those observed in generalist
scarabs are found in the social Hymenoptera (6, 11), where they
are characterized by distinct afferent modalities and Kenyon cell
populations. This organization suggests specialization of functions that may than be selectively integrated via combinatorial
outputs from the mushroom body lobes (11). Subcompartmentalization coincident with evolutionary expansion of higher brain
regions is also evident in mammals, as exemplified by the
elaborate functional subpartitions in the enlarged visual cortex
of primates (33). Regarding the third possibility, unique cognitive capabilities of honey bees, such as perception of generalized
and abstract rules about multiple stimuli in associative learning
paradigms (34, 35), suggest a role for the gyrencephalic mushroom bodies of these and perhaps other insects in enabling
complex learning and memory functions.
Sociality has been proposed to drive the evolution of large
higher brain centers in both insects and vertebrates (36, 37). In
insects, gyrencephalic calyces and large mushroom bodies are
observed in cockroaches, scarab beetles, and social hymenopterans. Cockroaches, although gregarious, are not considered social
at the level of ants, bees, and wasps. Elaborated mushroom body
lobes are found in the eusocial termites, but the calyces appear
to be reduced and fused relative to those of their sister taxon, the
17398 ! www.pnas.org"cgi"doi"10.1073"pnas.0508430102
cockroaches (38, 39). Similarly, gyrencephalic calyces are observed in basal nonsocial Hymenoptera such as sawflies (40).
Sociality, therefore, does not appear to be a robust predictor of
calyx morphology.
In scarabaeids, calyx morphology was uniformly associated
with feeding ecology. We propose that large gyrencephalic
calyces have evolved in response to selective pressure for behavioral flexibility that would be imparted by the above neural
properties and that would be beneficial to a generalist feeding
ecology in a long-lived mobile insect. For example, the adult
Japanese beetle Popillia japonica is long-lived (4–6 weeks) and
feeds on the tissues of %300 different host plant species (41),
which it locates by using olfactory cues such as host plant volatiles
induced by feeding damage (42) and visual cues (43). Despite this
wide host range, Popillia clearly prefers certain plant species
while rarely feeding on others (44), suggesting that these insects
are actively discriminating and choosing among many potential
food sources by using additional sensory cues. A long-lived insect
may also need to integrate this information with that regarding
food source handling, location, and availability, storing this
knowledge for future encounters. Cockroaches and social Hymenoptera are similarly long-lived and flexible in their feeding
behaviors and have arrived at a similar neural solution.
In contrast, dietary specialists using a few highly specific
cues to rapidly and unambiguously detect one or a closely
related group of host species would be expected to have fewer
requirements for sensory discrimination and integration and to
have a more hard-wired capacity for handling the host plant.
These insects would not be predicted to undergo such strong
selection for large mushroom bodies. Our results support this
prediction, and we suggest that the mushroom bodies are an
important component of the ‘‘larger and more sophisticated
nervous system’’ (45) that is a hypothesized requirement for
decision-making efficiency in insects with generalist feeding
ecologies (46).
The hypothesis that cognitive adaptations for generalist phytophagy played a key role in the evolution of gyrencephalic
calyces would equally predict that long-lived mobile generalist
feeders of any kind might display a similar evolutionary trend.
Generalist and specialist carnivorous beetles illustrate that this
is indeed the case (Fig. 4). The generalist predator Scarites
subterraneus possesses a gyrencephalic tripartite calyx capped by
a dorsally protruding Kenyon cell body region (Fig. 4A). In
contrast, two specialist predators, Scaphinotus elevatus (Fig. 4B)
and Harmonia axyridis (Fig. 4C), possess lissencephalic calyces
topped by smaller aggregates of Kenyon cell bodies.
Despite these robust results, feeding ecology is unlikely to be
equally predictive of mushroom body morphology across the
highly diverse insects. For example, it is unknown how quickly
changes in the brain can track changes in behavior. At the gross
Farris and Roberts
anatomical level, it is possible that an evolutionarily recent
switch in feeding ecology may not produce measurable differences in mushroom body morphology when compared with
related species retaining the ancestral feeding ecology. Developmental constraints may need to be overcome before significant enlargement or diminishment of the mushroom bodies
could occur. Additionally, other complex behaviors such as
social interaction and spatial navigation, also require advanced
capabilities for sensory integration, discrimination, learning, and
memory. Insects displaying these behaviors might be expected to
exhibit calyx gyrencephaly regardless of feeding ecology.
In vertebrates such as primates and birds, large and typically
gyri-rich cerebral cortices are correlated with the capacity for
behavioral innovation (particularly in terms of novel food source
usage) and success in invading novel environments (36, 47).
Polyphagous insects are also more likely to successfully establish
We thank Ms. Marissa Smith for assisting with data analysis,
Drs. Ronald Bayline and Kevin Daly for fruitful discussions, and
Drs. Susan Fahrbach and Gene Robinson for comments that greatly
improved this manuscript. N.S.R. was supported by Howard Hughes
Medical Institute Undergraduate Science Education Program Grant
52002683, awarded to Washington and Jefferson College. S.M.F. was
supported by West Virginia University.
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EVOLUTION
and maintain populations when colonizing new environments
(48). The worldwide ubiquity of cockroaches, social Hymenoptera, and phytophagous scarabs, many of which are economically
important invasive pests due to their colonization success, speaks
to a similar role for large gyrencephalic mushroom bodies.
Behavioral innovation is therefore likely to be a driving factor in
the evolution of large higher processing centers in the brains of
both invertebrates and vertebrates.
Farris and Roberts
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