Molecular Ecology (2006) 15, 3541– 3551

doi: 10.1111/j.1365-294X.2006.03030.x

Evolutionary diversification of the bean beetle genus
Callosobruchus (Coleoptera: Bruchidae): traits associatedwith stored-product pest status

Blackwell Publishing Ltd

M . T U D A ,* J . R Ö N N ,† S . B U R A N A P A N I C H P A N ,‡ N . W A S A N O §¶ and G . A R N Q V I S T †*Institute of Biological Control, Faculty of Agriculture, Kyushu University, Fukuoka 812-8581, Japan, †Animal Ecology, Departmentof Ecology and Evolution, Evolutionary Biology Centre, University of Uppsala, Norbyvägen 18d, SE-752 36 Uppsala, Sweden,‡Department of Entomology, Faculty of Agriculture, Chiang Mai University, Chiang Mai 50200, Thailand, §Biotechnology & Food
Research Institute, Fukuoka Industrial Technology Center, Aikawa, Kurume, Fukuoka 839-0861, Japan

Abstract
Despite the fact that many plant-feeding insects are pests, little effort has been made to
identify key evolutionary trait transitions that allow taxa to acquire or lose pest status. A
large proportion of species in the genus Callosobruchus are economically important pestsof stored, dry postharvest beans of the tribe Phaseoleae. However, the evolution of thisfeeding habit is poorly understood. Here, we present a reconstruction of the phylogeny of
the Asian and African Callosobruchus based on three mitochondrial genes, and assess
which traits have been associated with the evolutionary origin or loss of ability to reproduce on dry beans. Our phylogenetic analysis showed that species group into the chinensis
and the maculatus clades, which are also supported by genital morphology, and an additional paraphyletic group. Ancestral ability to use dry beans has been lost in the chinensis
clade but acquired again in C. chinensis. Dry-bean use and host-plant use were both phylogenetically constrained and transitions in the two were significantly correlated. Host
shifts from the subtribe Phaseolinae to Cajaninae were more common than the reverse andwere more likely in species using young beans. The ability to use dry beans was more likely
gained when using Phaseolinae hosts and promoted habitat shifts from tropical to temperate regions. Adaptation to arid climate was also associated with the ability to reproduce on
dry beans and on Phaseolinae. Thus, our analysis suggests that physiological adaptations
to an arid climate and to Phaseolinae hosts both render beetles predisposed to become pestsof cultivated beans.
Keywords: Bayesian inference, COI and COII, Fabaceae, host-plant shifts, plant–herbivore
interactions, Wolbachia
Received 16 February 2006; revision accepted 22 May 2006

Introduction
Comparative research on insect-plant co-evolution has
by tradition focused on parallel evolution in insects andtheir hosts, and has shown that herbivorous insects tendto specialize on specific plant taxa and/or plant tissues(Ehrlich & Raven 1964; Strong et al. 1984; Futuyma 1986;
Correspondence: M. Tuda, Fax: +81-92-642-3040; E-mail:tuda@grt.kyushu-u.ac.jp.
¶Present address: Insect Genetics and Evolution Department,
National Institute of Agrobiological Sciences, Tsukuba, Ibaraki
305-8634, Japan© 2006 The Authors
Journal compilation © 2006 Blackwell Publishing Ltd

Futuyma & Moreno 1988). Much less is known about thetypes of traits that allow insects to shift hosts and to utilize
particular resources. Our understanding of the evolutionof insect pests on agricultural crops is obviously an
important issue from an economic point of view, but may
also involve key adaptations that can shed general light on
the evolutionary ecology of herbivorous insects. Stored
product pests are a diverse group of insects that are well
adapted to using dry and hardened food as a resource(Robinson 1928; Chapman 1931; Linsley 1944; Ishii 1952;
Cotton 1956). While the hardness of dried seeds per se canserve as a deterrent against seed predators, including seedbeetles of the family Bruchidae (Janzen 1977; Southgate

3542 M . T U D A E T A L .
1979; Kitch et al. 1991; Dongre et al. 1993), the loss of toxic
chemicals during postmaturity drying processes may
also increase survival of such granivores. Bruchid beetles
primarily utilize beans from the family Fabaceae as theirhosts (Johnson 1981). Most bruchids are oligophagous;
their host range is limited to restricted plant taxa, typicallytribes and subtribes for species utilizing the legume
subfamily Faboideae (Jermy & Szentesi 2003; Tuda et al.
2005). The ability to use dry beans as a food resource iswidespread in this family. In Bruchinae, a derived groupwithin the Bruchidae, many nonpest species do exhibit theability to use dry seeds (Watanabe 1985; Shimada 1988;
Tuda et al., unpublished) and these may be pre-adapted to
becoming pests of stored beans (Watanabe 1985; Shimada
1990; Yoshida 1990).
The genus Callosobruchus Pic (Bruchinae) includes approximately 20 species (Borowiec 1987) and an unusually large
proportion of these are pests of stored beans. As larvae,
Callosobruchus species utilize seeds of legumes of the tribe
Phaseoleae (Fabaceae), including many beans cultivatedfor human consumption ( Johnson 1981). Recent researchon this genus has led to the discovery of several new
species that feed on legumes without economic importance (Arora 1977; Kingsolver 1999; Anton 2000; Tuda 2003;
Tuda et al. 2005). While one might think that it would benatural for taxa that utilize seeds of Phaseoleae to become
pests of stored beans, our understanding of why only somespecies are able to reproduce continuously, without intermission by reproductive diapause, on dry and hardenedbeans is very limited (see Labeyrie 1981). Climatic factors,

primarily the length of the reproductive season, have beensuggested as key correlates of pest status (Cotton 1956;
Kurota & Shimada 2002). However, other traits may also
play a role, such as the presence of endosymbiotic bacteria(Tsuchida et al. 2004).
Here, we seek associations between the evolution ofability to reproduce on dry beans and a suite of othercharacters in the genus Callosobruchus. We first test theextent to which the ability to use dry beans is phylogenetically constrained, and then test for associations with thefollowing variables: (i) host taxa in the wild; (ii) a suite ofclimatic variables; and (iii) the presence or absence ofendosymbiotic bacteria (i.e. Wolbachia).

Materials and methods
Taxon sampling
Mature leguminous seeds, both native and introduced,
were collected from various sites in tropical to temperate
Asia and Africa and were brought into the laboratory.
Emerging beetles were fixed and preserved in > 99%
acetone until use for molecular analysis. The sampledbruchids, their host plants, sampling location, andlocality of native distribution are listed in Table 1. We
used Zabrotes subfasciatus (Boheman) of the bruchid
subfamily Amblycerinae and Acanthoscelides obtectus (Say)of the subfamily Bruchinae as outgroups. A total of 14
Callosobruchus species were subjected to the following
analyses.

Table 1 Bruchid species, their host plants and locality of collection. Natural range estimation is shown in parentheses as Asian (A), African(Af) and American (Am)Bruchid species
Bruchinae
Callosobruchus analis (Fabricius)
Callosobruchus chinensis (Linnaeus)
Callosobruchus dolichosi (Gyllenhal)
Callosobruchus imitator Kingsolver
Callosobruchus latealbus (Pic)
Callosobruchus maculatus (Fabricius)
Callosobruchus nigripennis (Allard)
Callosobruchus phaseoli (Gyllenhal)
Callosobruchus pulcher (Pic)
Callosobruchus rhodesianus (Pic)
Callosobruchus semigriseus (Motsch.)
Callosobruchus subinnotatus (Pic)
Callosobruchus theobromae (Linnaeus)
Callosobruchus utidai Tuda
Acanthoscelides obtectus (Say)
Amblycerinae
Zabrotes subfasciatus (Boheman)

Host plant

Collector

Locality (natural range)

Vigna radiata
Vigna angularis
Cajanus scarabaeoides
Vigna umbellata
Rhynchosia acuminatifolia
Vigna unguiculata
Cajanus scarabaeoides
Lablab purpureus
Cajanus cajan
Vigna unguiculata
Dunbaria bella
Vigna subterranea
Cajanus scarabaeoides
Dunbara podocarpa
Phaseolus vulgaris

To
U
T
T
Ta
C
T
C
Ne
G
T
C
T
T
J+S

Myanmar (Af + A)
Japan (A)Thailand (A)Thailand (A)China (A)India (Af)Thailand (A)U.K. (Af)
Hawaii (A)Zimbabwe (Af)Thailand (A)Ghana (Af)Thailand (A)Thailand (A)Hungary (Am)

Phaseolus vulgaris

C

Columbia (Am)J + S, Jermy and Szentesi; T, Tuda et al.; Ta, Tateishi; U, Utida; To, Toquenaga; Ne, Nemoto; G, Giga; C, Credland.
© 2006 The Authors
Journal compilation © 2006 Blackwell Publishing Ltd

E V O L U T I O N O F C A L L O S O B R U C H U S S T O R E D - B E A N P E S T S 3543

DNA analysis
DNA was extracted with DNeasy kit (QIAGEN and
Machery-Nagel) following the manufacturers’ instructions. Partial sequences of mitochondrial genes, encoding
cytochrome c oxidase subunit I (COI) and subunit II(COII) that are interleaved by tRNA-Leucine (tRNALeu)
gene were amplified with the following primer sets:
5′-GGATCACCTGATATAGCATTYCC-3′ (modC1J-1751,
modified from Simon et al. 1994) in combination with
5′-GCTAATCATCTAAAAATTTTAATTCCTGTTGG-3′(revC1J-2441, modified from Simon et al. 1994), and5′-CTTTATCAACATTTATTTTGATTTTTT-3′ (COI2-1)
in combination with 5′-TACTCCAATAAATATTATAATAAATTG-3′ (COI2-2) for COI (Tuda et al. 1995, 2004); 5′TAATATGGCAGATTAGTGCATTGGA-3′ [Gomez-Zurita
et al. 2000; modified TL2-J-3037 (Simon et al. 1994)] and 5′GAGACCATTACTTGCTTTCAGTCATCT-3′ [Gomez-Zurita
et al. 2000; modified TK-N-3785 (Simon et al. 1994)] fortRNALeu — COII. Polymerase chain reaction and sequencing
were done as detailed in Tuda et al. (2004). The total size ofthe sequenced regions was 1651 bp (COI — 952 bp, tRNALeu— 21 bp and COII — 678 bp). The sequence data include no
indels and the base composition was AT rich, as is typicalfor insect mitochondrial sequences. The data are depositedin GenBank, under the accession nos DQ459035 – 459050 for
COI and DQ459020– 459034 for tRNALeu and COII.

Character states of host plants
Hosts were classified into either of the two subtribes ofPhaseoleae that were found to harbour Callosobruchus
beetles: Phaseolinae and Cajaninae. Primary host subtribeof respective beetle species was assigned according tohost taxa under nonstorage condition, by excluding all
records from stored beans (see Tuda et al. 2005). A recent
molecular phylogenetic study of the hosts (Kajita et al.
2001) showed that these two subtribes form strongly
supported groups and that Cajaninae is clearly monophyletic. The ability to use dry mature host beans as larval
medium was examined under natural as well as laboratoryenvironments for all bruchid species included here (Tudaet al., unpublished). We also checked for the presence ofWolbachia infections in multiple individuals of all species,
by using a pair of standard Wolbachia-specific primers(Kondo et al. in preparation).
Species habitats were classified into climatic zones basedon the data gathered from the online climate database World
Weather Information Service, provided by the World
Meteorological Organization (www.worldweather.org).
Since legumes in tropical and temperate areas bear seeds
in arid seasons, which provide bruchids resources toreproduce (Africa, Sanon et al. 1998; Delobel et al., unpublished; Asia, Niyomdham, personal communication; Tuda,© 2006 The Authors
Journal compilation © 2006 Blackwell Publishing Ltd

personal observation; Central America, Janzen 1967), we
focused on climatic variables during arid seasons. First,
the number of arid months (rainfall < 50 mm/month) andthe mean temperatures during the arid months in the
habitats of respective species (Appendix) were subjected
to a standard principal component analysis. The firstprincipal component explained a large fraction (93.6%)of variance in the climatic factors tested and the mean
temperature during the arid months correlated strongly
with this axis (r = 1.00) while the number of arid months
per year did not (r = – 0.0029). We hence classified habitats
into two groups, tropical and temperate, based on temperature (Table 2; Appendix I). Second, to test the effect ofaridity, we also categorized habitats into arid (arid months
per year ≥ 5 months) or humid (arid months per year
< 5 months) (Table 2; Appendix I).

Phylogenetic reconstruction
Model choice. Prior to phylogenetic analyses, a priori
selection of a nucleotide substitution model was done
based on the Akaike information criterion (Akaike 1973)
using the program mraic.pl version 1.4 (Nylander 2004) inconjunction with phyml version 2.4.4 (Guindon & Gascuel
2003). mraic.pl compares 24 standard substitution models,
including models allowing rate variation, by utilizing
likelihood scores calculated by phyml (all parameters
estimated during tree search). This analysis showed thata general time reversible model (GTR) (Rodriguez et al.
1990), combined with the assumption that a proportionof sites was invariable (I) (Gu et al. 1995) and a gamma
distribution for modelling rate variation (Γ) (Yang 1994),
minimized the Akaike information criterion for both COI
and COII. We hence chose this model (GTR + I + Γ) for our
entire data set.
Phylogenetic inference. We modelled our sequence data ina Bayesian framework, using mrbayes version 3.0b4 (seeHuelsenbeck & Ronquist 2001; Ronquist & Huelsenbeck2003). Exploratory analyses showed that the estimatedvalues for the model parameters were similar for COI and
COII, while the estimates differed substantially across thethree codon positions. We thus grouped the nucleotides at
the first, second, and third codon positions into different
partitions, and allowed the overall rate of substitution, thegamma shape parameter, the proportion of invariable
sites, and the rate of evolution (i.e. rate multipliers) todiffer at the different positions. This aside, we used default
priors for all model parameters.
The posterior distribution of trees was approximatedusing Markov chain Monte Carlo as implemented inmrbayes. We ran four independent runs with four parallel
chains (Metropolis coupling; Huelsenbeck & Ronquist
2001), where each run started from a randomly chosen

3544 M . T U D A E T A L .
Table 2 Host utilization, dry bean use and habitat climate of all species
Specieschinensis clade
C. chinensis
C. utidai
C. semigriseus
C. dolichosi
C. nigripennis
C. theobromae
C. pulcher
maculatus clade
C. analis
C. rhodesianus
C. maculatus
C. subinnotatus
Paraphyletic group
C. latealbus
C. imitator
C. phaseoli
Outgroup
A. obtectus
Z. subfasciatus

Natural hosts

Main host subtribe

Dry bean use

ClimateCajanus, Rhynchosia
Dunbaria, Vigna
Dunbaria
Dunbaria
Cajanus
Cajanus
Cajanus
Cajanus

CajaninaeYTe, arid

Cajaninae
Cajaninae
Cajaninae
Cajaninae
Cajaninae
Cajaninae

N
N
N
N
N
N

Tr, arid
Tr, humidTr, humidTr, humidTr, humidTr, humid

Vigna
Vigna
Vigna
Vigna

Phaseolinae
Phaseolinae
Phaseolinae
Phaseolinae

Y
Y
Y
Y

Tr, arid
Te, arid
Tr, arid
Tr, arid

Rhynchosia
Vigna
Lablab,Vigna

Cajaninae
Phaseolinae
Phaseolinae

YYYTe, humid
Tr, arid
Tr, arid

Phaseolus
Phaseolus

Phaseolinae
Phaseolinae

YYTe, arid
Tr, aridTe, temperate; Tr, tropical.

tree, with 10 million generations each and sampled every
1000th generation. The first 1 million generations were discarded in each run as a burn-in phase. A comparison of allparameters from the four runs, using tracer version 1.2.1(Rambaut & Drummond 2005), confirmed that the chains
had converged on the same target distribution and tree
topology in all runs (Huelsenbeck et al. 2002). Trees andparameter values from the four runs were then pooled anda majority-rule consensus tree was calculated.

Phylogenetic constraints and correlated evolution
We then used our majority-rule consensus molecularphylogeny to test evolutionary scenarios for the use of
dry beans in Callosobruchus. Congruence of dry bean use,host taxonomy, Wolbachia infection and climate with ourmolecular phylogeny was first tested by a permutation
tail probability (PTP) test (Faith & Cranston 1991) as
implemented in paup* (Swofford 2002), using 2000 replications. Randomization within characters was applied
only to ingroup taxa (i.e. Callosobruchus species) (Trueman
1996).
We tested for correlated evolution between all of ourvariables (including dry bean use) using a continuous-timeMarkov model in a maximum likelihood framework, as
implemented in discrete 4.0 (Pagel 1994). We used likelihood ratio test to compare independent- and dependent-

evolution models and their statistical significance wastested by Monte Carlo simulation with 2000 iterations,again using discrete 4.0. If the dependent evolution
model was significantly better than the independent
model, transition rates were further tested by setting thetransition rate to zero in the dependent model (i.e. abackward procedure). When backward procedure did
not detect any significant transition rates, alternatively, byallowing transition rates conditional to the state of theother trait (by adding a single transition rate to the independent model), we tested whether an additional transition rate was significant (i.e. a forward procedure). For
both backward and forward procedures, the critical valueof the likelihood ratio −2ln(H0/H1) is approximately χ 2
distributed with 1 degree of freedom (i.e. the difference inthe numbers of parameters between H0 and H1), where H0
and H1 are hierarchical models with the former being
the simpler (see Pagel 1994). We also tested whether eachtransition rate in the independent models was significantly
different from zero. We did not employ parsimony analysis
because of its tendency to underestimate evolutionarychange in long branches (Pagel 1999b).
When estimating ancestral states at internal nodes, weused a continuous time Markov model in a maximumlikelihood framework, using multistate (Pagel 1999a).
For optimization of ancestral states, we used local rather
than global optimization (see Pagel 1999b).
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E V O L U T I O N O F C A L L O S O B R U C H U S S T O R E D - B E A N P E S T S 3545

Results
Phylogenetic reconstruction
The Bayesian phylogenetic analysis yielded a well-resolved
phylogeny, given in Fig. 1, with high clade support, especially for more basal nodes (posterior probabilities 0.95–
1.00). The Bayesian posterior probability of this topology(with Callosobruchus dolichosi being sister species to thegroup containing C. semigriseus, C. utidai and C. chinensis)
was 0.199, and the 50% most credible set of topologies(cumulative posterior probability < 0.5) contained only
three additional topologies with only very minor alterations. In the first of these (posterior probability 0.156),
C. dolichosi and C. semigriseus group together as sister
species. In the second (posterior probability 0.055), C.
pulcher is a sister species to C. theobromae and C. nigripennis.
In the third (posterior probability 0.054), C. semigriseus
replaces C. dolichosi as the basal taxa to the other three
species in that clade.

Within the genus, three phylogenetic groups were thus
clearly distinguishable: one clade containing C. chinensis,
another containing C. maculatus and a third paraphyletic
group of species. This topology is also congruent withmorphological character states of male genitalia (seeTable 3). Species in the maculatus clade are primarily distributed in Africa and commonly feed on Vigna of the subtribe Phaseolinae (Tables 1 and 2, Fig. 1). The chinensis
clade primarily contains species from subtropical and/or
temperate Asia. The hosts of these species are Cajanus,Dunbaria and Rhynchosia of the subtribe Cajaninae, with
polyphagous species (i.e. C. chinensis) feeding on Vigna
as well (Table 2). The remaining paraphyletic group feedon either of two Phaseoleae subtribes under natural
conditions (Table 2).

Phylogenetic constraints and correlated evolution
The evolution of dry bean use and host utilization
were both significantly congruent with the Callosobruchus
Fig. 1 (a) molecular phylogeny of the genus
Callosobruchus. Shown is the 50% majorityrule consensus tree from the Bayesiananalyses. The numbers below nodes
represent Bayesian posterior probabilities.
Observed character states are indicated at
terminal nodes and the maximum likelihood
estimates of ancestral states at internal nodes
under local optimization, shown as pie
diagrams for probability of each character
state (left, the ability to use dry, hardened
mature beans; right, host subtribes). An
asterisk indicates that the probability of one
state at a given internal node is significantly
higher than that of the other.

© 2006 The Authors
Journal compilation © 2006 Blackwell Publishing Ltd

3546 M . T U D A E T A L .
Table 3 Morphology of male genitalia in Callosobruchus

Specieschinensis clade
C. chinensis
C. utidai
C. semigriseus
C. dolichosi
C. nigripennis
C. theobromae
C. pulcher
maculatus clade
C. analis
C. rhodesianus
C. maculatus
C. subinnotatus
Paraphyletic group
C. latealbus
C. imitator
C. phaseoli
Outgroup
A. obtectus

Host taxonomy
Wolbachia
ClimateTropical/Temperate

Median lobe

Lateral lobes (parameres)Endophallic
platesExophallic valve

long and slenderlong and slenderlong and slenderlong and slenderlong and slenderlong and slenderlong and slender

Y-shape, broad at apex
Y-shape, broad at apex
Y-shape, broad at apex
Y-shape, broad at apex
Y-shape, broad at apex
Y-shape, broad at apex
Y-shape, broad at apex

2
2
2
2
2
2
2

spear-head
spear-head
spear-head
spear-head
spear-head
spear-head
spear-head

short and stout
mediumshort and stout
short and stout

V-shape, broad at apex
V-shape, narrow at apex
V-shape, broad at apex
V-shape, broad at apex

2
6
0
2

triangulartriangulartriangulartriangularmediumlong and slender
mediumV-shape, broad at apex
V-shape, narrow at apex
V-shape, broad at apex

226

triangularspear-headtriangularmediumV-shape, broad at apex

2

triangular

Dry bean use

Host taxonomy

Wolbachia

3.88 (0.010)
2.32 (0.047)—
0.793 (0.28)——

2.92 (0.014)
4.25 (0.005)

0.599 (0.47)
5.39 (0.002)

1.62 (0.11)
0.758 (0.39)

phylogeny (PTP, P = 0.0045, P = 0.0045, respectively),while neither Wolbachia infection nor climatic variables werecongruent with the phylogeny (PTP, Wolbachia P = 1.0,
tropical/temperate P = 1.0, arid/humid P = 0.064).
We found a significant correlation in evolution of dry
bean use and host utilization (Table 4). The ancestral
state of the paraphyletic group and the maculatus clade wasability to use dry Phaseolinae seeds and the ancestral stateof the chinensis clade was utilization of young, fresh
Cajaninae seeds (Fig. 1). None of the transition rates weresignificantly different from zero, when tested by a backward procedure (–2 ln H0/H1 < χ12,0.05, P > 0.05). Whenassessed with a forward procedure, however, p34 wassignificantly higher than p12 (–2 ln H0/H1 = 7.95, P < 0.01),
indicating that a loss of the ability to use dry beans wasmore likely when the host is Cajaninae compared to when
it is Phaseolinae (see Fig. 2a for transitions). The opposite
trend was also significant. An ability to use dry beans wasmore likely to be gained when the host is Phaseolinae(p21 > p43) (–2 ln H0/H1 = 5.82, P < 0.025). Host shift wasalso conditional upon dry bean use, such that a shift from

Arid/Humid

Table 4 Likelihood ratio tests of correlated
evolution between dry-bean and hosttaxonomy, climate and Wolbachia infection.
Log likelihood ratios are followed by P
values in parentheses, estimated by Monte
Carlo simulations. Bolded P values indicate
significant differences at the critical level(P = 0.02)

Phaseolinae to Cajaninae was less likely to occur whenusing dry beans (p13 < p24) (–2 ln H0/H1 = 6.38, P < 0.025).
The independent evolution model of dry bean use
showed that both α1 (transition rate from young to drybean use) and β1 (transition rate from dry to young beanuse) were significantly different from zero and were 0.662
and 0.370, respectively. In the independent evolution
model of host subtribes used, the host shift rate from Phaseolinae to Cajaninae, β2, was significant (0.716) but thatfrom Cajaninae to Phaseolinae, α2, was not significantly
different from zero, indicating asymmetric host shifts
between the two host subtribes.
Transitions in dry bean use and tropical/temperate
climate were also correlated (Table 4) and the transition
rate q13 was significantly different from zero (–2 ln H0/
H1 = 7.60, P < 0.01, Fig. 2b), indicating that habitat shifts
from tropical regions to temperate regions are more likely
when using dry beans. No other transition rates qij weresignificantly different from zero (see Fig. 2b).
Transitions in dry bean use and the length of the arid season
were strongly correlated (Table 4). None of the transition© 2006 The Authors
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E V O L U T I O N O F C A L L O S O B R U C H U S S T O R E D - B E A N P E S T S 3547

Discussion

Fig. 2 Transition rates from models of correlated evolution ofcharacters in Callosobruchus. (a) Correlated evolution between dry
bean use and host subtribes. (b) Correlated evolution between dry
bean use and tropical/temperate climate. (c) Correlated evolution
between dry bean use and arid/humid climate. (d) Correlated
evolution between host subtribes and arid/humid climate. Thickarrows indicate transition rates that were significantly higher thaneither zero (by a backward procedure) or the rates indicatedby thin arrows (by a forward procedure). Broken-line arrows
are transition rates that were nonsignificant from zero. *: P < 0.05,**: P < 0.01, ***: P < 0.001.

rates were significantly different from zero, when tested
with a backward procedure (–2 ln H0/H1 < χ12,0.05, P > 0.05).
However, with a forward procedure, beetles using dry bean
were more likely to shift from humid to arid areas thanwere beetles using young beans (r31 > r42), whereas beetles
using young beans were more likely to shift from arid tohumid areas compared to those using dry beans (r24 > r13)
(Fig. 2c). Arid habitat also promoted an evolutionary shift
to the use of dry beans (r21 > r43) whereas humid habitats
was associated with the reverse shift (r34 > r12) (Fig. 2c).
Transitions in host utilization and shifts between arid/
humid habitats were significantly correlated (Table 4).
Forward tests showed that the use of Phaseolinae wasmore likely in arid climate and use of Cajaninae was more
likely in humid areas (see Fig. 2d).
Infections by Wolbachia was only marginally correlatedwith dry bean use and uncorrelated with the other traits(Table 4).
© 2006 The Authors
Journal compilation © 2006 Blackwell Publishing Ltd

In general, our results suggest that there are two stable
adaptive zones of evolutionary attraction within the genus
Callosobruchus and that transitions between these areunlikely. One is to occupy humid areas with short arid
season and use young and fresh beans of Cajaninae hosts.
The other is to inhabit arid environments with longer arid
season and use Phaseolinae hosts. It is species exhibiting
the latter characteristics that show the ability to use dry
and hardened beans as a food resource and thus tend to
become pests of stored products. Below, we will discuss
some of the implications of our findings.
Our analyses suggest that the use of Cajaninae as hostplants may in effect be an evolutionary trap in Callosobruchus.
Species using Cajaninae (with the exception of C. chinensis)
do not use Phaseolinae even though Vigna are abundant inmany of the areas occupied by these species (Tuda et al.,unpublished). In contrast, C. maculatus and other Phaseolinaeusing species are capable of utilizing some Cajaninae species
as well, at least in the laboratory (Janzen 1977; Dongre et al.
1993). The dry seeds of Cajaninae may have a thicker coat
(Dongre et al. 1993) and generally seem harder to penetrate
than those of Phaseolinae, and this may prevent species in
the chinensis clade from evolving an ability to use the dryseed stages of their hosts. Similarly, the hardness of dry
Cajaninae beans may limit the natural host range of species
utilizing dry or mature stages of Phaseolinae beans.
The use of dry beans may also have promoted a shift
from tropical habitats to temperate regions. Adaptation to
dry food, either through use of metabolic water stored inbound form or through diapause, can indirectly enhancecold hardiness (Robinson 1927; Chapman 1931; Block 1996)
and may thereby be associated with an ability to tolerate
lower temperatures. Adaptation by bruchids to low humidity
in arid areas may have further served as pre-adaptation to
the use of seeds with low water content (i.e. dry beans).
Conversely, adaptation to a humid climate may prevent
bruchids from developing physiological tolerance against
low water content of dry seeds. Alternatively, long arid
seasons and the associated availability of legume seeds may
tend to favour repeated reproduction, or multivoltinism,in bruchids. In the process of losing reproductive diapause,difficulty in lengthening adult lifespan under aridity may
have contributed to gaining reproductive ability without
feeding (Watanabe 1990). There is, however, at least one
exception to this generalization. C. latealbus inhabits humid
areas but is able to feed on dry host seeds. This may be due
to its basal position in the genus or, alternatively, to infections by Wolbachia. For the latter, careful experimental
studies are needed to elucidate the potential importance ofsuch symbionts.
Recent comparative studies on bruchid beetles haveconcluded that this group shows fairly high taxonomic

3548 M . T U D A E T A L .
conservatism in host use, but that host shifts to distant taxa
do occur during their evolution (Kergoat et al. 2004, 2005a,b). The results of our study agree well with this general
conclusion. Major Callosobruchus clades specialize on particular host subtribes and while host shifts to different taxa
are rare they do occur. A host-selection experiment using
the two subtribal hosts (Vigna vs. Cajanus) shows evolutionary lability of adult preference (behavioural adaptation) and evolutionary conservatism in larval performance(physiological adaptation) in C. maculatus (Wasserman &Futuyma 1981), and the latter could help maintain hostspecificity (see also Credland 1987; Tucic et al. 1995; forintrageneric host selection, see Kawecki & Mery 2003;
Messina 2004). Our study also highlights the uniquenessof C. chinensis within the chinensis clade. This species
apparently shows adaptations that allow it to utilize a wide
range of maturing stages of host beans and to have a wide
natural host range and a large geographical distribution(Tuda et al. 2005). The nature of the adaptations that provide C. chinensis these capabilities requires further investigation, including its interaction with Wolbachia and thefactors discussed below.
For bruchids pre-adapted to utilize dry seeds, proximity
to human stores of dried seeds (Cotton 1956) and polymorphism in dispersal and reproductive traits (Utida 1954,
1967, 1972, 1981; Kiritani 1955; Caswell 1960; Nakamura
1966; Sano 1967; Ouedraogo & Huignard 1981; Messina
1984; Sano-Fujii 1984, 1986; Umeya 1987) should havefurther promoted evolution towards gaining pest status.
The level of secondary metabolites in beans decreases
during the process of drying and the stores of manypotential host beans in close proximity may have contributed to a decreased level of host selectivity by adults,
thus resulting in the widening of host range seen in, forexample, C. chinensis and C. maculatus.
Janzen (1969; 1981) stressed morphology, phenology andthe chemistry of seeds as the most important factors determining interactions between bruchids and their host plants.
Secondary metabolites, such as nonprotein amino acids,
can certainly serve as chemical defence against herbivores
including granivores (Birch et al. 1989; Gatehouse et al. 1990).
We note that Phaseoleae species lack a toxic secondary
compound, l-canavanine, observed in other Faboideae(Rosenthal 1977; Bell et al. 1978; Bisby et al. 1994), which could
help explain the fact that the host range of Callosobruchus
is limited to Phaseoleae. In support of this suggestion,bioassay tests have shown lethal effects of canavanine on
the larvae of C. maculatus (Janzen et al. 1977; Oliveira et al. 1999).
Likewise, it is possible that chemical defence substances
may limit the host range of the seed predators of monophyletic Cajaninae (cf. Dongre et al. 1993). Furthermore,morphological traits of hosts, such as the size and textureof beans, can play an important role as proximate cues forhost recognition by adult beetles (e.g. in C. chinensis, Ishii

1952; in C. maculatus, Sulehrie et al. 2003; in other bruchine,
Szentesi 2003).
In summary, our study indicates that adaptation to along arid climate tends to make potential pest species
more likely to gain stored-bean pest status. However, ourresults also show that idiosyncrasies of particular taxa,
such as specific adaptations to their natural hosts, play animportant role.

Acknowledgements
This paper is dedicated to late Emer. Prof Syunro Utida whose
discussion on life history polymorphism of stored bean pest
Callosobruchus stimulated this work. The authors thank H.R. Pajni,
P. Tewari and K.-W. Anton for identification of Callosobruchus
theobromae and Callosobruchus semigriseus, M. Shimada, K. Fujii,
A. Szentesi, T. Jermy, and Y. Toquenaga for providing some of
the bruchid specimens, N. Kondo for providing unpublisheddata, F. J. Messina and G. J. Kergoat for valuable comments, and
N. Berti, H. Perrin (MNHN, Paris) for access to museum bruchidcollections. J.A.A. Nylander provided invaluable advice on Bayesian
phylogenetic analysis. This study was supported by the Fujiwara
Natural History Foundation, the Sumitomo Foundation, Zoologiska
Stiftelsen, the Swedish Research Council, and Grant-in-Aids forInternational Scientific Research (Field Research 09041145) and for
Scientific Research (A) (08304049, 15208007) (B) (14405003, 17405005)
and for Young Scientists (B) (15770011) from MEXT of Japan.

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MT pursues evolutionary ecology of plant-herbivore andherbivore-predator (or parasitoid) interactions, using pest insects
and their natural enemy. JR and GA are interested in sexual
selection and male-female coevolution. SB’s interest lies inagricultural entomology in general. NW is working on microbial
control of insect pests by using Bacillus thuringiensis.

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E V O L U T I O N O F C A L L O S O B R U C H U S S T O R E D - B E A N P E S T S 3551

Appendix I

Clustering of species’ habitats according to the mean temperature (°C) and the number of arid months (rainfall < 50 mm/month) per year.
Solid line circle: temperate; dashed line circle: tropical; solid line square: arid; dashed line square: humid. Habitat codes for the different
species are; c, chinensis; pu, pulcher; a, analis; r, rhodesianus; m, maculatus; s, subinnotatus; l, latealbus; ph, phaseoli; i, imitator; u, utidai; A, A.
obtectus; Z, Z. subfasciatus; and N, Northern Thailand (semigriseus, dolichosi, nigripennis, theobromae).

Appendix II
Aridity and temperature of the habitats of Callosobruchus species. As a typical habitat, we considered a location in which a species wasabundant and frequently observed or, alternatively, where the type specimen was collected. Aridity was measured as the number of months
with low rainfall (< 50 mm/month) per year

Specieschinensis clade
C. chinensis
C. utidai
C. semigriseus
C. dolichosi
C. nigripennis
C. theobromae
C. pulcher
maculatus clade
C. analis
C. rhodesianus
C. maculatus
C. subinnotatus
Paraphyletic group
C. latealbus
C. imitator
C. phaseoli
Outgroup
A. obtectus
Z. subfasciatus

Habitat location

Months withlow rainfall

Mean temperature (°C)
during the arid months

Kunming, China
Patna, India
Chiang Mai, Thailand
Chiang Mai, Thailand
Chiang Mai, Thailand
Chiang Mai, Thailand
Manila, Philippines

6
7
4
4
4
4
4

11.4
23.0
23.6
23.6
23.6
23.6
28.0

Port Louis, Mauritius
Harare, Zimbabwe
Abuja, Nigeria
Kumasi, Ghana

7
7
5
5

24.2
16.7
27.9
27.3

Guiyang, China
Mae Hong Son, Thailand
Mahajanga, Madagascar

4
5
6

8.2
24.0
25.6

Mexico City, Mexico
Los Cabos, Mexico

6
10

15.5
22.7

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