Long-Term Evolutionary Stability of Bacterial Endosymbiosis in
Curculionoidea: Additional Evidence of Symbiont Replacement in the
Dryophthoridae Family
Cyrille Conord,* Laurence Despres,* Agnès Vallier, Séverine Balmand, Christian Miquel,*
Stéphanie Zundel,* Guy Lemperiere,* and Abdelaziz Heddi *Laboratoire d’Écologie Alpine, Centre National de la Recherche Scientifique Unité Mixte de Recherche 5553, Université Joseph
Fourier, Grenoble, France; and UMR203 Biologie Fonctionnelle Insectes et Interactions, Institut Fédératif de Recherche41, Institut
National de la Recherche Agronomique, INSA-Lyon, Villeurbanne, France
Bacterial intracellular symbiosis (endosymbiosis) is well documented in the insect world where it is believed to play
a crucial role in adaptation and evolution. However, although Coleopteran insects are of huge ecological and economical
interest, endosymbiont molecular analysis is limited to the Dryophthoridae family. Here, we have analyzed the
intracellular symbiotic bacteria in 2 Hylobius species belonging to the Molytinae subfamily (Curculionoidea superfamily)
that exhibit different features from the Dryophthoridae insects in terms of their ecology and geographical spanning.
Fluorescence in situ hybridization has shown that both Hylobius species harbor rod-shaped pleiomorphic symbiotic
bacteria in the oocyte and in the bacteria-bearing organ (the bacteriome), with a shape and location similar to those of the
Dryophthoridae bacteriome. Phylogenetic analysis of the 16S ribosomal DNA gene sequences, using the heterogeneous
model of DNA evolution, has placed the Hylobius spp. endosymbionts (H-group) at the basal position of the ancestral Rclade of Dryophthoridae endosymbionts named Candidatus Nardonella but relatively distant from the S-clade of
Sitophilus spp. endosymbionts. Endosymbionts from the H-group and the R-clade evolved more quickly compared with
free-living enteric bacteria and endosymbionts from the S- and D-clades of Dryophthoridae. They are AT biased (58.3%
A þ T), and they exhibit AT-rich insertions at the same position as previously described in the Candidatus Nardonella
16S rDNA sequence. Moreover, the host phylogenetic tree based on the mitochondrial COI gene was shown to be highly
congruent with the H-group and the R-clade, the divergence of which was estimated to be around 125 MYA. These new
molecular data show that endosymbiosis is old in Curculionids, going back at least to the common ancestor of Molytinae
and Dryophthoridae, and is evolutionary stable, except in 2 Dryophthoridae clades, providing additional and independent
supplementary evidence for endosymbiont replacement in these taxa.
Introduction
Associations between insects and bacteria are common in nature and include a wide range of interactions from
parasitism to mutualism. In insects subsisting on nutritionally deficient habitats, symbiotic associations may occur
and involve intracellular bacteria (endosymbionts) living
within specialized host cells (the bacteriocytes) that sometimes form an organ, the bacteriome (Buchner 1965). Endosymbionts are thought to provide limiting components to
insects (Douglas 1998), thus improving their fitness and their
invasive power (Heddi et al. 1999). They could also be involved in insect temperature resistance (Montllor et al.
2002), protection against parasites (Oliver et al. 2003), or
broadening of food plant range (Tsuchida et al. 2004). Endosymbionts often transmit vertically to insect offspring for hundreds of generations, resulting in cospeciation between the
bacteria and the insect (Baumann et al. 1995). As a result, this
route of symbiont transmission leads to a peculiar symbiont
DNA evolution. The most striking features are the tendency
to genome size reduction (Shigenobu et al. 2000; Gil et al.
2002; Nakabachi etal. 2006; Pérez-Brocal et al. 2006),a global
enrichment in AT nucleic bases (Moran 1996; Heddi et al.
1998) and fast nucleotide substitution rates (Moran 1996).
Molecular phylogenetic studies have provided a broad
insight into the evolutionary history of many insect group
endosymbioses, including aphids (Moran et al. 1993), carKey words: endosymbiosis, evolution, phylogeny, Molytinae,
Dryophthoridae, Curculionoidea.
E-mail: [email protected].
Mol. Biol. Evol. 25(5):859–868. 2008
doi:10.1093/molbev/msn027
Advance Access publication February 29, 2008
Ó The Author 2008. Published by Oxford University Press on behalf of
the Society for Molecular Biology and Evolution. All rights reserved.
For permissions, please e-mail: [email protected]
penter ants (Schröder et al. 1996), tsetse flies (Chen XA et al.
1999), psyllids (Thao et al. 2000), weevils (Lefèvre et al.
2004), mealybugs (Downie and Gullan 2005), and leafhoppers (Takiya et al. 2006). Cospeciation appears as the
dominant pattern of evolution between the primary
endosymbiont and the host lineages, and the stability of
the association is so strong that, in insects harboring multiple endosymbionts, multiple cocladogenesis has been
shown to occur in some cases (Takiya et al. 2006). However, recent molecular data have indicated that associations
with a given endosymbiont are not necessarily persistent
and might have often been lost and replaced by different
microbes during insect evolution. Such symbiont replacements have been suspected in insect groups wherein multiple
bacterial types are coexisting within a single host lineage
(Moran and Baumann 1994; Gomez-Valero et al. 2004).
Such a replacement event could be governed by a switch
of the host habitat and may result from the competition between primary endosymbionts with a reduced exhausted genome and secondary endosymbionts that express a beneficial
role for host fitness (Chen DQ et al. 2000; Sabater et al. 2001;
Montllor et al. 2002; Koga et al. 2003; Gomez-Valero et al.
2004; Lefèvre et al. 2004; Tsuchida et al. 2004).
The Curculionoidea superfamily is the largest group of
the most species-rich order of living organisms, the Coleoptera. There are more than 60,000 species of weevils
worldwide that may be found in a variety of habitats, ranging from seashores to mountain tops and from deserts to
rainforests, where they are particularly abundant and diversified (Zimmerman 1993). These insects have succeeded in
thriving on a large range of plant structures, such as roots,
stems, branches, flower heads, fruits, and seeds of both angiosperms and gymnosperms. Some are pests of major
860 Conord et al.
concern to agriculture and forestry, and others are beneficially used in the biological control of noxious weeds.
However, in spite of their ecological and geographical importance, very little is known about intracellular symbiosis’
evolution and its role in weevil adaptation. So far, only the
Dryophthoridae weevils—that are mainly distributed in
tropical regions and also represent important pest species
in temperate regions—have been studied with modern microbiological and molecular techniques (Lefèvre et al.
2004). In this family, 3 clades of bacterial symbionts that
differ in their evolutionary rates and their nucleotide composition have been defined. The D-clade (53.4% Guanine/
Cytosine content) includes endosymbionts of Diocalandra
frumenti and Trigonotarsus rugosus; the S-clade (53.8%
GC content) comprises the endosymbionts of Sitophilus rugicollis, Sitophilus granarius, Sitophilus zeamais, and Sitophilus oryzae; and the R-clade (40.5% GC content) includes
the endosymbionts of Yuccaborus frontalis, Rhynchophorus palmarum, Cosmopolites sordidus, Sphenophorus abbreviata, Scyphophorus yuccae, Metamasius hemipterus,
and Metamasius callizona. All these Dryophthoridae belong to the Rhynchophorinae subfamily, except Y. frontalis
that belongs to the Orthognathinae. The R-clade, which
includes the endosymbionts of the most ancestral Dryophthorid insect analyzed (i.e., Y. frontalis), is the oldest clade
that was established 100 MYA (Lefèvre et al. 2004). The Sand D-clade endosymbionts have become associated more
recently, probably by symbiont displacement.
To expand our knowledge on endosymbiosis occurrence and evolution in the Curculionoidea and test the hypothesis of evolutionary succession of endosymbionts in
the Dryophthoridae family, we have investigated endosymbiosis in 2 species of the Molytinae subfamily, Hylobius
abietis and Hylobius transversovittatus. Molytinae members are widely distributed in the northern and southern
hemispheres. Furthermore, most Hylobius weevils, including H. abietis, are restricted to conifer trees and breed in the
roots of dying or recently dead conifers. Hylobius transversovittatus insects are among the few exceptions that breed
in the lignified roots of the perennial plant Lythrum salicaria, and they are used as a biological control agent of the
invasive host plant in North America (McAvoy et al. 2002).
We show that both Hylobius species harbor pleiomorphic endosymbiotic bacteria in a very conserved bacteriome
structure, similar to that of the Dryophthoridae. Host and
endosymbiont phylogenetic tree construction revealed
a strong concordance between the endosymbionts and their
hosts from Molytinae and Dryophthoridae and indicated
that Molytinae and their endosymbionts branched with
the Dryophthoridae insects and endosymbionts (R-clade),
respectively. We conclude that Curculionoidea endosymbiotic associations are older than previously estimated for the
Dryophthoridae insects and that symbiont displacement
may have occurred at least twice in the S- and D-clades
of the Dryophthoridae.
Materials and Methods
Insect Collection and Bacterial DNA Extraction
Insects were collected in the field and kept alive on
host plant organs until dissection. Hylobius abietis and
H. transversovittatus were collected in France, on Pinus
pinaster (Ait.) in a clear-cut forest site (44°01#58$N and
1°14#41$W) and on L. salicaria (L.) in a meadow next
to a wetland (45°05#45$N and 5°47#31$E), respectively.
Ovaries and larval bacteriomes were dissected in
buffer A (25 mM KCl, 10 mM MgCl2, 250 mM sucrose,
and 35 mM Tris–HCl, pH 7.5) and homogenized in 250 ll
of buffer STE (Sodium Chloride, Tris, EDTA: 100 mM
NaCl, Tris–HCl, pH 8, and 1 mM Na2-ethylenediaminetetraacetic acid [EDTA]), and Tris–Hcl, pH 8). DNA was
extracted by adding 3 ll of proteinase K (18 mg/ml) and
then incubating for 2 h at 55 °C. RNA contamination
was removed with the addition of 3 ll of RNaseA
(10 mg/ml) for 1 h at 37 °C. DNA was then purified with
phenol–chloroform extraction and precipitated with 0.7 v/v
of isoamelic alcohol. For insect DNA extraction purposes,
imago flight muscles were dissected in buffer A and DNA
was extracted using a DNeasy Tissue Kit (Qiagen, Hilden,
Germany), following the instructions of the manufacturer.
Bacterial 16S rDNA Polymerase Chain Reaction
Amplification and Sequencing
Because we had no prior indications regarding bacterial phylogenetic origin, bacterial 16S rDNA amplification
was performed using eubacterial universal primers: 008 forward 5#-AGAGTTTGATCMTGGCTCAG-3# and 1,389
reverse 5#-GACGGGCGGTGTGTACAA-3# (nucleotides
numbering from Escherichia coli, GenBank accession
number J01859). Reactions were carried out in a volume
of 50 ll consisting of 2.6 units of Taq DNA polymerase
(Roche, Basel, Switzerland), 1.75 mM MgCl2, 0.35 mM
deoxynucleoside triphosphate (dNTP), 0.3 lM primers,
and 10 ng of DNA template. The polymerase chain reaction
(PCR) parameters were as previously described (Lefèvre
et al. 2004). Sequencing was performed on PCR products
by Genome Express (Grenoble, France), according to the
Applied Biosystems protocol, on an automatic sequencer
(ABI Prism 3100).
Fluorescence In Situ Hybridization Procedure
Larval bacteriomes, apexes of the ovaries, and mature
oocytes were dissected in buffer A and squashed on poly-Llysine-coated microscope slides. Slides were heated for
5 min on a hot plate at 60 °C and then treated for 5 min with
acetic acid (70%), at 45 °C, in a moisture chamber. They
were rinsed for 5 min with phosphate buffer saline (PBS,
pH 7.2), dehydrated in different alcohol solutions, and
digested with 100 lg/ll pepsin in 0.01 N HCl for 10 min
at 37 °C. Larval bacteriomes were fixed during 1 week
in Finefix solution (Milestone, Bergamo, Italy) and embedded in paraffin. Five micrometer thick sections were
mounted on poly-L-lysine-coated microscope slides. After
methylcyclohexane dewaxing and rehydration, sections
were digested with 100 lg/ll pepsin in 0.01 N HCl for
10 min at 37 °C.
The specific oligonucleotide probe used was designed
by sequence alignment of Hylobius endosymbionts 16S
rDNA (5#-ACCCCCCTCTATGAGAC-3#) and 5# end
labeled with rhodamine. Hybridization was performed at
Endosymbiosis in the Curculionoidea
45 °C in a dark moisture chamber in a 0.9 M NaCl, 20 mM
Tris–HCl, 5 mM EDTA, 0.1% sodium dodecyl sulfate, and
10 Denhardt solution. After a 30-min preincubation period, 25 ng/ll of probe were added and incubation
continued for 3 h. Slides were washed twice in the same
buffer at 45 °C for 15 min, rinsed with PBS, and mounted
in Gel Mount (Biomeda, Foster City, CA) medium containing 4#,6-diamidino-2-phenylindole (DAPI). Tissue imaging was carried out on Zeiss microscope (Axiovert). To
confirm the specific detection of the endosymbionts, several
control experiments were undertaken including Rnase digestion negative control, signal-decrease control with excess of unlabeled probe, no-probe control, and the use of
a Wolbachia 16rDNA probe (Heddi et al. 1999).
Phylogenetic Analyses and Topology Test for Bacteria
Endosymbiont DNA is characterized by evolutionary
rates significantly higher and a genomic GC content that is
generally lower, when compared with closely related freeliving bacteria (Heddi et al. 1998). The among-site rate variation and base composition heterogeneity challenge the
standard reconstruction of phylogenetic trees, particularly
when organisms are highly AT biased and have evolved
at relatively high rates. Hence, the long AT-rich branches
of insect endosymbionts have always been difficult to place
in the phylogenetic tree with free-living relatives (Lefèvre
et al. 2004; Herbeck et al. 2005). Therefore, phylogenetic
trees were constructed using the heterogeneous model of
DNA sequence evolution developed by Galtier and Gouy
(1998; Galtier et al. 1999) implemented in the nhml software (ftp://pbil.univ-lyon1.fr/pub/mol_phylogeny/nhml).
This model implements both variations of substitution rates
among sites and unequal base composition across lineages
and provides, as output, the estimated equilibrium GC content of each sequence (i.e., the GC content, it would have if
it were to evolve indefinitely with the evolutionary bias of
its terminal node) and the estimated GC contents in ancestral sequences.
The 16S rDNA sequences alignment, provided as Supplementary Material online by Lefèvre et al. (2004), was
used as a reference. Sequences of Hylobius endosymbionts
were added, and a new alignment was performed using
ClustalW, implemented under BioEdit (Hall 1999), followed by manual refinement. Regions of ambiguous alignment were removed from all analyses, resulting in a data set
of 1,388 bp in total.
Alternative topologies were compared using the exploration method developed by Lefèvre et al. (2004) for
a similar data set. This method involved taking the a priori
fixed topology of weevil endosymbionts and free-living
bacteria published in Lefèvre et al. (2004) and adding Hylobius endosymbionts, either one by one or together, on
each branch of the reference topology. For each tested endosymbiont position, the tree log likelihood was estimated.
A bootstrap was performed on the 16S rDNA data set (500
replicates) in order to test for the final phylogenetic tree.
Alternative topologies were compared using t-tests on their
log-likelihood scores (500 values for each topology). In
addition, the internal structure of the final tree was tested with
local ‘‘pruning regrafting’’ procedures implemented in the
861
NHML software (Non-homogenous maximum likelihood for
phylogenetics: ftp://pbil.univ-lyon1.fr/pub/mol_phylogeny/
nhml). Only the subtree formed by the endosymbionts of the
R-clade and the branches of the Hylobius endosymbionts were
allowed to be rearranged. Finally, to assess confidence interval
(CI) on the most likely tree, we performed an approximately
unbiased (AU) test using CONSEL 1.19 (Shimodaira and
Hasegawa 2001). Sitewise log likelihoods under this model
were computed using nhPHYML (Boussau and Gouy 2006)
and were used as input for the tests implemented in CONSEL
with default scaling and replicate values. The topology obtained by Lefèvre et al. (2004) using the heterogeneous model
of Galtier and Gouy (1998) was again used as a backbone on
which all possible positions of Hylobius endosymbionts were
added, yielding 63 topologies. Additionally, to evaluate the
likelihood of weevil endosymbiont monophyly, a subset of
the permutation of the 4 clades was tested given that
S,[R,D] was found to be the most likely topology when
Dryophthoridae endosymbionts were tested alone (Lefèvre C,
personal communication), that is, S,[D,[R,H]] versus
H,[S,[R,D]] versus S,[H,[R,D]] versus S,[R,[D,H] allowed
tomoveonthetreebackboneformedexclusivelywithfreebacteria. Then, the most likely topologies under hypothesis of
monophyly (designated by the AU test) were compared with
topologies constructed under a hypothesis of polyphyly, that
is, D with Haemophilus/Pasteurella clade plus [R,S] branched
on the enteroclusters and with the H-group allowed to move
on every node versus H,[R,D] with the S-clade allowed to
move on every node.
Relative Rate Tests
The substitution rates of the 16S rDNA gene were
compared between endosymbionts of the Hylobius clade
and the Dryophthoridae clades and with several representatives of free-living bacteria and other insect endosymbionts, using the RRTree software (Robinson and
Huchon 2000) and Aeromonas haywardensis (AF015258)
as an outgroup. The null hypothesis was that the rate of substitution of the tested clade is the same as that of the reference group.
Insect COI PCR Amplification and Sequencing
Two specific primers were designed for Hylobius COI
mtDNA amplification: 76 forward 5#-CGGAATAGTTGGTACATC-3# and 1,278 reverse 5#-AATCCTAAGAAATGTTGAG-3’ (nucleotides numbering from Drosophila
yakuba GenBank accession number X03240). Reactions
were carried out in 25 ll containing 1 unit of Taq DNA
polymerase (Ampligold, Roche), 2 mM MgCl2, 0.2 mM
of each dNTP, 1 lM primers, and 2 ll of DNA template.
The PCR amplification reaction included an initial 10-min
denaturing step at 95 °C followed by 40 cycles at 95 °C for
30 s, 48 °C for 30 s, 72 °C for 1 min, and a final elongation
step at 72 °C for 7 min. COI mtDNA sequencing was performed on an ABI 3100 capillary automatic sequencer using the big dye terminator sequencing Kit V3.1 from
Qiagen.
Nucleotide sequences were deposited in the GenBank
database, and the other endosymbiont and weevil sequences
862 Conord et al.
used were obtained from the same database (GenBank accession numbers given in figs. 2 and 3, respectively).
Phylogenetic Analysis of Host–Insect Relationships
Two outgroup taxa were chosen within Curculionoidea.
Lepidapion cretaceum was randomly selected among the
COI sequences of Apionidae available in Genbank (accession
number AJ717656), and Tanysphyrus lemnae was chosen
within Erirhinidae (DQ768215). In addition, another
Molytinae species was added, namely Pissodes strobi
(AY131096), a North American species living on conifers,
inwhichnoendosymbioticbacteriahavesofarbeenobserved.
A total of 16 sequences were aligned using ClustalW
and all regions with ambiguous alignment were excluded,
resulting in a total alignment of 1,246 bp. Parsimony analysis revealed a high rate of homoplasy (CI 5 0.43), which
is not surprising given the available gene used (i.e., COI
mtDNA) and the presumably high level of divergence between the studied taxa. Therefore, nucleotide sequences
were translated into amino acid sequences using the appropriate reading frame. This considerably reduced homoplasy
(CI 5 0.68), allowing for a more reliable phylogenetic reconstruction. A phylogenetic analysis was performed on
this data set using the substitution model MtRev for mitochondrial proteins implemented in PHYML (Guindon and
Gascuel 2003).
Statistical Test of Cospeciation
A statistical test of cospeciation between the weevils and
their symbiotic bacteria was performed using the program
ParaFit (Legendre et al. 2002). ParaFit statistically assesses
the fit between host and symbiont phylogenetic distance matrices mediated by the matrix of host–symbiont links. Matrices of patristic distances were built from both endosymbiont
and host–insects phylogenies. Each matrix in turn was then
transformed into a matrix of principal coordinates that was
used as input for ParaFit. Probability for the test of cospeciation was computed after 999 permutations.
Results
Endosymbiosis in Hylobius Species
Symbiotic bacteria were histologically examined in
the oocytes, the apexes of ovarioles, and the larval bacteriome of both H. transversovittatus and H. abietis. These
species were shown to harbor intracellular bacteria that permanently infect the oocyte and the apex of ovarioles, as described in Dryophthoridae species (Nardon et al. 2002). The
Hylobius larval bacteriome is also of a similar shape and
location as the bacteriome of Dryophthoridae insects
(Scheinert 1933; Buchner 1965). It surrounds the foregut/midgut junction as a single, ring-shaped structure consisting of numerous lobelets of different sizes but without
any communication with the gut (fig. 1B and C). A fluorescence in situ hybridization (FISH) experiment was conducted, with a specific probe, in order to confirm that the
16S rDNA sequences, generated by PCR from H. transversovittatus and H. abietis bacteriomes, were derived from the
endosymbionts we had observed. Endosymbiotic bacteria
appear to be pleiomorphic in all the samples examined
(fig. 1). They are rod shaped and their size varies from
1.5 to 50 lm, depending on the insect stage and tissue. Although in the oocyte, bacteria are short rodlets that measure
from 1.5 to 5 lm, they increase in size in the bacteriomes
and become filamentous, from 6 to 30 lm in the ovariole
bacteriome and up to 50 lm in the larval bacteriome.
Phylogenetic Molecular Analyses of the 16S rDNA
Sequence from Hylobius Endosymbionts
Phylogenetic Tree Construction
Prior to the phylogenetic analysis using the heterogeneous model of evolution, distance, maximum likelihood
and maximum parsimony analyses were performed. In
all these analyses, high bootstrap values supported the basal
position of Hylobius endosymbiont sequences to the
R-clade of the Dryophthoridae (see Supplementary Material
online).
The Hylobius endosymbiont sequences were highly
AT biased and exhibited AT-rich insertions in the same positions, as previously described in the bacterial R-clade of
Dryophthoridae by Lefèvre et al. (2004) and in Buchnera
endosymbionts (Lambert and Moran 1998). Three AT-rich
insertions were found to be common to the R- and Hylobius
clades and absent in all other endosymbionts analyzed
(including the 16S sequences of primary endosymbionts
from 10 aphids, 3 whiteflies, and 7 carpenter ant species
and weevils ES from S- and D-clades): positions
150–200, 215–245, 885–895, and 1085–1095 (see Supplementary Material online). These 3 AT-rich regions are synapomorphies that support the monophyly of ES from R- and
H-clades and exclude the S- and D-clades and therefore
strongly supporting the hypothesis of symbiont replacement in the Sitophilus species: R-, S-, and D-clades clearly
are not monophyletic. The GC contents were 41.7% and
42.1% for the Hylobius clade and for the Dryophthoridae
R-clade, respectively.
When tested alone, each of the Hylobius bacterial sequences branched together with the R-clade. When tested
simultaneously, the highest likelihood tree grouped the
Hylobius endosymbionts together with the R-clade subtree
(position 51 on the reference tree, see fig. 2). Among the
205 trees evaluated by the shake procedure, the highest likelihood topology was the same as the one obtained by the
topology exploration method. Statistical comparisons of
the topologies confirmed this topology as the most significant (node 51; log-likelihood score: 7727.23; t-test,
P , 1.104). This was consistent with the best tree found
by the shake procedure when branches of the R-clade and
the 2 Hylobius endosymbionts were allowed to be rearranged. The final topology is given in figure 3. The name
of ‘‘H-group’’ was assigned to the group defined by the
2 Hylobius endosymbiont sequences.
Monophyly versus Polyphyly of the Weevil
Endosymbionts
When sequences of the H-group were allowed to move
freely on the tree backbone, the tree with the highest
Endosymbiosis in the Curculionoidea
863
FIG. 1.—Bacteriome morphology and FISH of Hylobius endosymbionts in different tissues. Panel (A) shows the structure of isolated larval
bacteriome lobelets from the foregut/midgut junction. Panels (B) and (C) are FISH experiments on bacteriome sections; numerous lobelets around the
intestine (B) and the intimate contact of the bacteriome with the intestine (C) are shown. Panels (D–F) are FISH squashes from oocytes, ovariole
bacteriomes, and larval bacteriomes, respectively. Slides (B–F) were hybridized with a specific probe designed on Hylobius 16S rDNA and stained with
DAPI. N: nucleus; E: endosymbiont; bl: bacteriome lobelets; I: intestine; and T: tracheal.
likelihood among the 63 possible topologies corresponded
to a position of the Hylobius sequences basal to the whole
R-clade. The topology with H sequences grouped with Yuccaborus had a lower likelihood but was not significantly
different, whereas all other topologies were significantly
less likely (threshold: P . 0.05: positions 51 and 53, see
fig. 3 and table 1A).
Under the hypothesis of monophyly, the topology with
the highest likelihood corresponded to the tree where
S,[D,[R,H]] branched with the Haemophilus/Pasteurella
clade (table 1B, threshold: P . 0.05).
The global comparison of polyphyletic versus
monophyletic topologies for the weevil endosymbiont
clades showed unambiguously that the tree, where H se-
quences are grouped with R sequences, is significantly
the likely than all other topologies (table 1C, threshold:
P . 0.05).
Relative Rates of Evolution of Hylobius Endosymbionts
The H-group showed rates of evolution significantly
different from all other bacterial clades, except the R-clade
(table 2). Therefore, as it has been deduced for sequences of
the R-clade of Dryophthoridae, the Hylobius endosymbionts evolved more rapidly than both free-living enteric
bacteria and the endosymbiotic bacteria from the S- and
D-clades. Thus, we define an ‘‘Rh-group’’ comprising
R-clade and H-group sequences, to be used further in the
864 Conord et al.
ples, P 5 0.009). However, a detailed examination of
the results showed that the test was significant only for
the host–symbiont couples of the S-clade and the H-group.
In a second step, when performing a test on the R-clade and
the H-group only, the global test was highly significant
again (9 insect–bacteria couples, P 5 0.001), but this time
all the pairwise tests of cospeciation were significant, that
is, for the R-clade and the H-group taxa.
Discussion
Endosymbiosis Occurrence in the Curculionoidea
Superfamily
FIG. 2.—Log likelihood for 63 tree topologies for alternative placement
of the 2 Hylobius species’ endosymbionts. Log-likelihood values are plotted
relative to the highest among them (position 51 in the topology). Ha and Ht
stand for Hylobius abietis and Hylobius transversovittatus‘ endosymbionts
16S sequences, respectively; Ha and Ht designate a simultaneous test of the 2
sequences across the topology positions.
text. Given an estimated rate of evolution of 0.128 substitutions per site per 100 Myr for the R-clade (Lefèvre et al.
2004), the H-group would have diverged approximately
115 MYA.
Host–Insect Phylogenetic Analysis
The amino acid alignment showed 73 parsimony informative characters out of 415. The tree obtained by the
maximum likelihood analysis is given in figure 3. Molytinae weevils were grouped together with Dryophthoridae
with a high bootstrap support. This topology was also
strongly supported by distance analysis (data not shown).
The molecular clock hypothesis was not verified on
the whole weevil phylogeny, that is, COI substitution rates
were not constant among the different weevil lineages (likelihood ratio test P 5 3.5 105). However, relative rate
tests performed with RRTree, using the 2 outgroups as reference sequences, showed no significant difference in sequence evolution between Dryophthoridae and Molytinae
(P 5 0.76). Given that the oldest known Molytinae fossil
is ;55-Myr old (Britton 1960; Zherikhin 2000), the divergence between Dryophthoridae and Molytinae must have
taken place earlier. Accepting an age of 100 Myr for the
Dryophthoridae group (O’Meara 2001; Lefèvre et al.
2004) gives an estimate for the divergence between the
Dryophthoridae and the Molytinae between 125 and 165
MYA, that is, between the mid-Jurassic and the end of
the late Cretaceous period.
It is well recognized and documented that insects are
the most diverse group of macroscopic organisms.
Throughout the world, some 900,000 different kinds of living insects are known, which approximates to 80% of the
world’s species. According to Erwin (1983), the number of
living species of insects has been estimated to be 30 million.
Out of this huge diversity, only a few (less than one thousand) have been studied with regards to intracellular symbiosis, which greatly underestimates the extent of symbiosis
within this large animal group. Among approximately 30
orders of insects, Coleoptera is the largest order in the entire
animal kingdom. There are about 350,000 named species of
beetles in the world and many more unnamed species
(Chadwick 1998). So far, less than 20 species, all belonging
to the Dryophthoridae family, have been investigated molecularly and were shown to harbor intracellular bacteria.
Here, we have extended the modern microbiological characterization of bacterial endosymbionts in the Curculionoidea superfamily beyond the Dryophthoridae group. We
show that those Molytinae species also harbor bacteria related
to Candidatus Nardonella described in Lefèvre et al. (2004).
Interestingly, endosymbiont examination in both Hylobius
species has revealed that the bacteriome structure is histologically similar to the structure observed in the Dryophthoridae,
which supports a deep-seated and long-term relationship between the Curculionoidea and their endosymbionts. Furthermore, Hylobius exhibits similar pleiomorphism as in the
Dryophthoridae, with a relatively small shape in the oocytes
that becomes bigger and filamentous in both the larval and
ovariole bacteriome. Scheinert (1933) hypothesized that endosymbiotic bacteria might undergo a process of ‘‘degeneration’’ during their passage from the larval stage of the host to
the adult sexual cells. The FISH study showed that the bacterialstrainobservedin bacteriomesandin oocytesisthe same
despite the significant morphological modifications. Along
with the previous observation in the Dryophthoridae, this
finding suggests that bacterial pleiomorphism is of a host–
symbiont interaction origin and that beetles might use a conserved molecular mechanism to control endosymbiont cell
division and growth, presumably through the inhibition of
bacterial cell septation during the insect development.
Cospeciation between the Hosts and the Endosymbionts
Endosymbiosis Evolution within the Curculionoidea
Superfamily
In a first step, the global test of cospeciation performed
with ParaFit on trees comprising all weevils and their endosymbionts was highly significant (13 insect–bacteria cou-
The host–insect and endosymbiont trees were strongly
congruent for the Rh-group as certified by the cocladogenesis tests, supporting a scenario of cospeciation between
Endosymbiosis in the Curculionoidea
865
FIG. 3.—Maximum likelihood phylogenetic tree of Hylobius and Dryophthoridae endosymbionts and maximum likelihood phylogram of host–
insects based on COI amino acid sequences. (ES) indicates endosymbiotic bacteria; other taxa are free-living bacteria. The marked node (number 51)
corresponds to the most likely position of the Hylobius clade on the tree topology. For the insect-hosts tree values on the branches are bootstrap values
(1,000 replicates). Bootstrap values of less than 70% are not reported.
host–insects and their endosymbionts from an ancestor
common to the Hylobius species and the Dryophthoridae
and a single bacterial infection. Positive signal of global cospeciation between the complete set of bacteria and their
host–weevils was obtained with a separate treatment of
the S-clade and the Rh-group. This indicated that a cospeciation scenario is supported both within the S-clade and the
Rh-group but that another evolutionary event had to occur
to explain the global pattern observed. This scenario leads
to the following evolutionary arguments on the weevil
endosymbiosis.
First, the age of endosymbiosis in weevils could be at
least 25 Myr older than previously estimated from the Dryophthoridae group alone (;100 Myr, Lefèvre et al. 2004).
Given an age of divergence of 125 Myr for weevil endosymbionts, the estimation of the 16S rDNA evolutionary
rate is 0.125 substitutions per site per 100 Myr (s/s/100
Myr), very close to the rate estimated for the R-clade alone
(0.128 s/s/100 Myr, Lefèvre et al. 2004) and higher than
those calculated for Buchnera (0.058 s/s/100 Myr), as well as
for the free-living enterobacteria (0.007–0.018 s/s/100 Myr,
Clark et al. 1999), but lower than those estimated for
866 Conord et al.
Table 1
Rank
1
2
3
14
27
28
Branching node
51
53
40
9
37
48
Tree topology
H-group basal to R-clade
H-group within R-clade
H-group with S-clade
H-group with D-clade
H-group basal to R- and S-clades
H-group within Enterobacteria
D lnL
6.6
6.6
115.7
160.5
845.8
851.6
2
4
6
3
B
Rank
1
5
70
71
Branching node
5
5
4
4
Tree topology
S,[D,[R,H]]
H,[S,[R,D]]
S,[H,[R,D]]
S,[R,[D,H]]
D lnL
0.8
18.8
98.4
98.4
AU
0.724
0.152
3 10048
2 10064
C
Rank
1
2
7
Hypothesis
Polyphyly D5[R,S]8 þ H
Polyphyly D5[R,S]8 þ H
Monophyly S,[D,[R,H]]
D lnL
3.9
3.9
50.4
AU
0.834
0.309
0.002
25
polyphyly [H,[R,D]]5 þ S
Branching node
H branches at the basis of R
H branches with Yuccaborus
Weevil clade branches with Haemophilus/Pasteurella
S-clade branches outside the (Haemophilus,
Pasteurella,[H,[R,D]]) clade
123.8
2 10066
A
AU
0.912
0.088
10005
10037
10008
10024
NOTE.—Comparison of alternative topologies of 16S sequences of Hylobius endosymbionts. The topologies were tested with CONSEL (Shimodaira and Hasegawa
2001). The first column indicates the rank of the topologies given their log likelihood. The second column indicates the branching node of the moving group of sequences as
numerated on figure 3 and described in the third column. D lnL: log-likelihood difference. AU: approximately unbiased test. A: exploration of the 63 placements of the Hgroup. The highest likelihoods for positioning of the H-group across the different clades are detailed. B: global comparison of 128 topologies under constrained monophyly
for the 4 weevil endosymbiont clades. C: global comparison of 144 topologies under constrained polyphyly or monophyly for the weevil endosymbiont clades.
the carpenter ant endosymbiont Blochmannia (0.24 s/s/100
Myr, Degnan et al. 2004).
Second, symbiont inheritance across such a large time
span indicates strong benefits for the insect host. The biological significance of endosymbiosis has not been addressed in
Hylobius weevils so far. However, given the low digestibility
and the weak nutritional value of the deadwood diet, which is
supposed to be the ancestral habitat of weevils (Marvaldi
et al. 2002), early association with bacteria able to provide
complementary nutrients might have represented a key innovation in this group of insects. In addition to chemical and
nutritional characteristics, the nature and the availability
in space and time of the Molytinae diet resources might
act as a constraint on the biology and behavior of the insect.
Recent studies have supported a high dispersal ability in
H. abietis (Nilssen 1984; Conord et al. 2006), similar to many
other Curculionoidea insects. This particularity might have
evolved because the adult is feeding on young conifer seedlings, while the larva develops in the root cambial tissues of
freshly dead conifers, which could be considered as a scarce
and ephemeral resource. Furthermore, most suitable host
plant tissues are exhausted when the adult emerges so that
the new generation must migrate and find new breeding sites.
This life cycle requires that insects fly from their feeding sites
to their egg laying and developmental sites, which could be
considerably distant and, hence, energy consuming. Regarding this aspect, it was noteworthy that Sitophilus endosymbionts were shown to highly increase insect mitochondrial
energetic metabolism (Heddi et al. 1993, 1999) and to improve the adult flight ability (Grenier and Nardon 1994).
Hence, it is likely that endosymbiosis might also impact
on the flight performance of other weevils and enhance their
dispersal ability and invasive power.
Finally, this work has provided additional support for
the occurrence of bacterial replacement in Dryophthoridae.
In addition to the molecular evidence discussed by Lefèvre
et al. (2004), the branching of the Hylobius endosymbionts
within the R-clade and relatively distant from the S-clade
has confirmed the polyphyly of the Dryophthoridae endosymbionts and therefore sustains symbiont displacement in
Sitophilus weevils. However, the factors favoring symbiont
replacement, either internal (weevil physiology) or external
(ecological influences), remain speculative. Whether a host
plant shift was favored by symbiont replacement or, on the
other hand, whether habitat shift has increased the probability that a new symbiont acquired horizontally (or a
secondary endosymbiont) outcompetes the ancestral endosymbiont is still an open question. Seed-eating species of
the Molytinae have been described, which could provide
a means of testing for an eventual link between endosymbiont replacement and the change in the nature of the weevil’s diet (Lyal 1996; Vanin and Gaiger 2005). This could
be addressed by exploring endosymbiosis in monophyletic
groups where drastic host shifts have likely occurred.
Table 2
Tested Clade
Hylobius
Hylobius
Hylobius
Hylobius
Hylobius
Hylobius
Hylobius
Rh-clade
Rh-clade
Rh-clade
clade
clade
clade
clade
clade
clade
clade
Reference Clade
P Value
R-clade
S-clade
D-clade
Enterobacteriaceae
Camponotus floridanus
Schizaphis graminum
Glossina pallidipes
Enterobacteriaceae
S-clade
D-clade
0.07
107
107
107
107
3.105
107
107
107
107
NOTE.—Evolutionary rates of the 16s rDNA sequences in the weevils’
endosymbionts tested with RRTree with Aeromonas haywardensis as outgroup. Rh
designates the clade formed by the R-clade of Dryophthoridae endosymbionts and
the Hylobius endosymbionts (see fig. 3 and text).
Endosymbiosis in the Curculionoidea
In conclusion, this work has opened new evolutionary
and symbiotic perspectives in the Curculionoidea, the largest insect superfamily in terms of species number. Moreover, endosymbiosis in the Curculionoidea may have
been established before the divergence between the Dryophthoridae and the Molytinae, at least 125 MYA. This
strongly argues in favor of a long-term stability of symbiosis in the Curculionoidea. Finally, symbiosis stability does
not mean symbiont persistence and continuity as endosymbiosis in the Curculionoidea may have evolved several
times through symbiont competition and displacement.
Updating the taxonomic status of ‘‘Candidatus Nardonella endosymbionts’’: Proposition of ‘‘Candidatus Nardonella dryophthoridicola’’ as the taxonomic name of the
R-clade endosymbionts and of ‘‘Candidatus Nardonella
hylobii’’ as the taxonomic name of the Rh-clade endosymbionts, according to the rules published in Murray and
Stackebrandt (1995).
In their paper, Lefèvre et al. (2004) proposed the generic name Candidatus Nardonella for the symbiotic bacteria of the R-clade. Given the similarities for several traits
between the R- and Rh-clade endosymbionts (e.g., similarities in shape and position of the larval bacteriome, in shape
and size pleiomorphism of the bacteria, and nucleic synapomorphies on the 16S rDNA sequences described in the
present paper), we suggest that the Rh-clade endosymbionts
will be included within the genus Nardonella. To distinguish between the R- and Rh-clades, we further suggest
different species names: Candidatus Nardonella dryophthoridicola name will replace ‘‘Candidatus Nardonella’’
name previously attributed to the R-clade endosymbiont
in Lefèvre et al. (2004). The Candidatus Nardonella dryophthoridicola describes hence the Dryophthoridae R-clade
endosymbionts strictly.
Candidatus Nardonella hylobii name will be used to design the Hylobius Rh-clade endosymbionts. The description
of Candidatus Nardonella hylobii is as follows: phylogenetic
position, c3 subclasses of Proteobacteria; cultivation, not
cultivated on cell-free media; morphology, rod shape, pleiomorphic size from 1.5 to 50 lm in length, 1–2 lm in diameter; basis of assignment, 16S rDNA sequences; association
and host, intracellular symbionts of Molytinae weevils
(described from oocytes, ovariole, and larval bacteriomes
in H. abietis and H. transversovittatus); mesophilic; authors,
Conord et al. (this study). The generic name honors Professor
Paul Nardon (Lefèvre et al. 2004), and the species name
is based on the genus name of the 2 weevil host species.
Supplementary Material
Supplementary figures are available at Molecular Biology and Evolution online (http://www.mbe.
oxfordjournals.org/).
Acknowledgments
The authors would like to thank 2 anonymous reviewers for their help in the improvement of the paper. They
also thank R. Douzet for his help with Lythrum salicaria,
F. Nicolè and M. Quivrin for their help with the weevil col-
867
lections, B. Boussau for advices on phylogenetic analyses,
and V. James and D. Wainhouse for the manuscript reading
and the English correction. This work was partly supported
by the French Ministère de la Recherche.
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Accepted January 20, 2008