# Endosymbiont Phylogenesis in the Dryophthoridae Weevils: Evidence for Bacterial Replacement

Cédric Lefèvre, Hubert Charles, Agnès Vallier, Bernard Delobel, Brian Farrell, Abdelaziz Heddi

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Cédric Lefèvre, Hubert Charles, Agnès Vallier, Bernard Delobel, Brian Farrell, et al.. Endosymbiont Phylogenesis in the Dryophthoridae Weevils: Evidence for Bacterial Replacement. Molecular Biology and Evolution, 2004, 21 (6), pp.965 - 973. 10.1093/molbev/msh063. hal-03504074

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# Endosymbiont Phylogenesis in the Dryophthoridae Weevils: Evidence for Bacterial Replacement

Cédric Lefèvre,* Hubert Charles,* Agnès Vallier,* Bernard Delobel,* Brian Farrell,† and Abdelaziz Heddi*

*Laboratoire de Biologie Fonctionnelle Insectes et Interactions, UMR INRA/INSA de Lyon, Villeurbanne, France; and

†Museum of Comparative Zoology, Harvard University

Intracellular symbiosis is widespread in the insect world where it plays an important role in evolution and adaptation. The weevil family Dryophthoridae (Curculionoidea) is of particular interest in intracellular symbiosis evolution with regard to the great economical and ecological features of these invasive insects, and the potential for comparative studies across a wide range of host plants and environments. Here, we have analyzed the intracellular symbiotic bacteria of 19 Dryophthoridae species collected worldwide, representing a wide range of plant species and tissues. All except one (*Sitophilus linearis*) harbor symbiotic bacteria within specialized cells (the bacteriocytes) assembled as an organ, the bacteriome. Phylogenetic analysis of the 16S rDNA gene sequence of the Dryophthoridae endosymbionts revealed three endosymbiotic clades belonging to γ3-Proteobacteria and characterized by different GC contents and evolutionary rate. The genus name *Candidatus* Nardonella was proposed for the ancestral clade infesting Dryophthoridae 100 MYA and represented by five of nine bacterial genera studied. For this clade showing low GC content (40.5% GC) and high evolutionary rate (0.128 substitutions/site per 100 Myr), a single infection and subsequent cospeciation of the host and the endosymbionts was observed. In the two other insect lineage endosymbionts, with relatively high GC content (53.4% and 53.8% GC), competition with ancestral pathogenic bacteria might have occurred, leading to endosymbiont replacement in present-day last insects.

## Introduction

Symbiotic associations are widespread through the
biotic world and occur at different levels of organismal
complexity, ranging from bacterial associations (von
Dohlen et al. 2001) to animal and plant symbioses
(Nardon and Grenier 1991; Margulis 1993). The formed
unit involves at least two separate species and may include
several organisms belonging to distant phyla. These
interspecific associations are believed to improve the
partner's fitness through integrated endosymbiosis (Mar-
gulis 1993). Endosymbionts may also induce reproductive
isolation and subsequent host speciation, such as in the
*Wolbachia* endosymbioses (Werren 1997; Bordenstein,
O'Hara, and Werren 2001). However, forces driving the
establishment and evolution of symbioses are still poorly
understood, and they probably vary from group to group.
The early steps in symbiogenesis are often believed to
be parasitic (environmental bacteria that possess infection
capacities), during which time, the partners exchange
virulence gene products, leading to selection at the pop-
ulation level (Yamamura 1996; Heddi 2003).

In insects subsisting on nutrient-deficient or un-
balanced diets, such as blood (Rhodnius, Glossina), plant
sap (aphids, psyllids, whiteflies, mealybugs), and cereal
grains (Sitophilus), symbiosis involves intracellular bacte-
ria (endosymbionts) living within specialized host cells
termed bacteriocytes, which sometimes form an organ, the
bacteriome (Buchner 1965). Intracellular bacteria, through
their interaction with the bacteriocytes, are generally
thought to supplement their host's diet with limiting
nutrients (Wicker 1983; Douglas 1998; Heddi et al. 1999).
Nevertheless, intracellular symbionts are not limited to

insects with unbalanced diets, but could also be involved
in insect temperature resistance (Montllor, Maxmen, and
Purcell 2002), host-plant detoxification (Dowd 1991), and
parasite protection (Olivier et al. 2003).

Apparent symbiont-host coevolution is known or
suspected in several systems where symbionts (living
within bacteriocytes) are always transmitted vertically
through female germ cells to the offspring (Baumann et al.
1995). Indeed, most of the insect associations studied so
far involve monophyletic lineages of intracellular bacteria,
whose phylogenesis follows host divergence (Munson
et al. 1991; Bandi et al. 1995; Schröder et al. 1996; Chen,
Li, and Aksoy 1999). Host-symbiont phylogeny con-
cordance strongly implies a single bacterial infection of
the host ancestor and subsequent cospeciation between
the bacteria and the insects. Endosymbiont genome evo-
lution is submitted to intracellular life-style constraints
(i.e., maternal mode of transmission with population bottle-
necks, bacteria insulation within bacteriocytes, and relaxed
selection) and is characterized by evolutionary rates sig-
nificantly higher and genomic GC content generally low
when compared to closely related free-living bacteria
(Brynnel et al. 1998; Heddi et al. 1998). This concordance
is due to elevated mutation rate and biases, lack of re-
combination, and the fixation of deleterious mutations by
genetic drift (Moran 1996) (for review see Wernegreen
[2002]). Among-site rate variation and base composition
heterogeneity challenge the reconstruction of phylogenetic
trees, particularly when organisms are highly AT-biased
and have evolved at relatively high rates (Loomis and
Smith 1990; Hasegawa et al. 1993). Hence, the long AT-
rich branches of insect endosymbionts have always been
difficult to place in the phylogenetic tree with free-living
relatives (Charles, Heddi, and Rahbé 2001).

The weevil family Dryophthoridae, which comprises
about 140 genera, is of a particular interest with regard to
intracellular symbiosis evolution. Most Dryophthoridae

Key words: intracellular symbiosis, insect, molecular phylogeny, GC content, evolutionary rate, evolution.

E-mail: abdelaziz.heddi@insa-lyon.fr.

Mol. Biol. Evol. 21(6):965-973. 2004

DOI:10.1093/molbev/msh063

Advance Access publication January 22, 2004
<table><caption>**Table 1**
Symbiotic Status of *Dryophthoridae* Weevils</caption><thead><tr><th>Species</th><th>Origin DNA<br>Template PCR</th><th>Plant Host<br>(organ and tissue)</th><th>Sample Origin</th><th>First Endosymbiont<br>Description</th></tr></thead><tbody><tr><td>Sitophilus granarius</td><td>LB, OB</td><td>Poaceae (seed)</td><td>France</td><td>(Mansour 1930)<sup>c</sup></td></tr><tr><td>Sitophilus oryzae</td><td>LB, OB</td><td>Poaceae (seed)</td><td>France</td><td>(Pierantoni 1927)</td></tr><tr><td>Sitophilus zeamais</td><td>LB, OB</td><td>Poaceae (seed)</td><td>France</td><td>(Musgrave and Homan 1962)</td></tr><tr><td>Sitophilus vateriae</td><td>MCB<sup>a</sup></td><td>Dipterocarpaceae (seed)</td><td>India</td><td>(Nardon et al. 2002)</td></tr><tr><td>Sitophilus linearis</td><td>Asymbiotic</td><td>Fabaceae (seed)</td><td>Senegal</td><td>(Nardon et al. 2002)</td></tr><tr><td>Sitophilus rugicollis</td><td>LB, OB</td><td>Myrtaceae (seed)</td><td>India</td><td>This work<sup>c</sup></td></tr><tr><td>Yuccaborus frontalis</td><td>LB</td><td>Agavaceae (stem)</td><td>Mexico</td><td>This work<sup>c</sup></td></tr><tr><td>Sphenophorus abbreviata</td><td>OB</td><td>Cyperaceae (root)</td><td>France</td><td>This work<sup>c</sup></td></tr><tr><td>Cosmopolites sordidus</td><td>LB, OB</td><td>Musaceae (root, stem)</td><td>Guadeloupe</td><td>(Nardon et al. 1985)<sup>c</sup></td></tr><tr><td>Trigonotarsus rugosus</td><td>LB</td><td>Xanthorrhoeaceae (trunk)</td><td>Australia</td><td>(Nardon et al. 2002)<sup>c</sup></td></tr><tr><td>Rhynchophorus ferrugineus</td><td>LB</td><td>Arecaceae (stem)</td><td>Cathar</td><td>(Buchner 1965)</td></tr><tr><td>Rhynchophorus palmarum</td><td>LB, OB</td><td>Arecaceae (stem)</td><td>Guadeloupe</td><td>(Nardon et al. 1985)<sup>c</sup></td></tr><tr><td>Metamasius hemipterus</td><td>LB, OB</td><td>Polymorphic (stem)</td><td>Guadeloupe</td><td>(Nardon et al. 1985)<sup>c</sup></td></tr><tr><td>Metamasius callizona</td><td>LB, OB</td><td>Bromeliaceae (stem)</td><td>USA</td><td>This work<sup>c</sup></td></tr><tr><td>Diocalandra frumenti</td><td>LB</td><td>Arecaceae (stem)</td><td>Australia</td><td>(Nardon et al. 2002)<sup>c</sup></td></tr><tr><td>Sipalinus gigas</td><td>LB<sup>b</sup></td><td>Polymorphic (dead wood)</td><td>Australia</td><td>(Nardon et al. 2002)</td></tr><tr><td>Scyphophorus yuccae</td><td>OB</td><td>Liliaceae (stem)</td><td>USA</td><td>This work<sup>c</sup></td></tr><tr><td>Dynamis borassi</td><td>LB<sup>b</sup></td><td>Arecaceae (stem)</td><td>Brazil</td><td>(Nardon et al. 2002)</td></tr></tbody></table>

LB: larval bacteriome; OB: ovarian bacteriome; MCB: mesenteric caeca bacteriome.

<sup>a</sup> In *Sitophilus vateriae*, a bacteriome-like tissue was observed at the apex of the mesenteric caeca, but no positive PCR have been obtained on the tissue yet (work in progress).

<sup>b</sup> Bacteriome-tissue was unambiguously observed on larvae, but no positive PCR amplification was obtained probably because of DNA template damage.

<sup>c</sup> 16S rDNA sequencing in this work.

are restricted to feeding on monocotyledonous angiosperms and feed on leaves, stipes, and roots, while known exceptions include *Sipalinus* and *Trigonotarsus*, which feed on decaying wood, and *Sitophilus*, which feed on seeds, including some dicots (Delobel and Grenier 1993). To date, few Dryophthoridae species in four genera have documented endosymbiotic bacteria. These include *Sitophilus oryzae* (L.) (Pierantoni 1927; Nardon and Grenier 1988), *Sitophilus granarius* (L.) (Mansour 1930), *Sitophilus zeamais* (Mot.) (Musgrave and Homan 1962), *Cosmopolites sordidus* (Germar), *Metamasius hemipterus* (L.) (Nardon et al. 1985), and *Rhynchophorus ferrugineus* (Buchner 1965). In all these species, intra-cellular bacteria were shown to occur in the bacteriome organ and in the oocytes (Nardon et al. 2002).

The present work greatly broadens the known incidence of endosymbiosis occurrence to some 13 genera within the Dryophthoridae through concerted molecular and histological surveys of species collected worldwide, and it provides initial evidence of multiple origins and replacement of their endosymbionts.

## Material and Methods

### Insect Collections

Experiments were conducted with species reared in the laboratory (i.e., *S. oryzae*, *S. zeamais*, *S. granarius*) and with species collected from the field worldwide (table 1). Collected species were sent either alive or immersed in 70% alcohol or fixative fluid BMN (20 ml of 1.5% picric acid solution in 80% ethanol, 5 ml of 40% neutral formalin solution, 2 ml of glacial acetic acid, and 23 ml H₂O).

## Total DNA Extraction

For living insects, ovaries or bacteriomes were dissected in buffer A (25 mM KCl, 10 mM MgCl₂, 250 mM sucrose, 35 mM Tris-HCl, pH 7.5) and DNA extractions were performed on the symbiont-bearing organs. For fixed insects, bacteriome tissues were manually collected from toluidine blue-strained sections with a thin needle under the microscope. DNA was then extracted using the Nucleon HT Kit (Amersham, Life Science).

## 16S rDNA Polymerase Chain Reaction (PCR) Amplification and Sequencing

The primers used for 16S rDNA amplification were the eubacterial universal primers: 008for 5'-AGAGTTTG-GATCMTGGCTCAG-3' (nucleotides 8-27, *Escherichia coli* numbering GenBank accession no. J01859) and 1389rev 5'-GACGGGCGGTGTGTACAA-3' (nucleotides 1,389-1,406). Reaction mixtures consisted of 2.6 units of Taq DNA polymerase and proofreading polymerase (Expand Long Template PCR System, Roche), 1.75 mM MgCl₂, 0.35 mM deoxynucleotide triphosphate, 0.3 µM primers, and 10 ng of DNA template in a final volume of 50 µl. The PCR parameters (Biometra T-gradient, Biolabo) were 94°C for 2 min followed by three series of cycles: (1) four cycles of 94°C for 10 s, 51°C to 54°C (0.5°C increment each cycle) 30 s, and 68°C for 30 s; (2) six cycles of 94°C for 10 s, 54°C for 30 s, and 68°C for 30 s; (3) 20 cycles of 94°C for 10 s, 54°C for 30 s, and 20 s more each cycle, and 68°C for 30 s. 16S rDNA sequencing was performed on PCR products by Genome Express (Grenoble, France) according to the Applied Biosystems protocol on an automatic sequencer.

Ten new sequences of endosymbionts were obtained from 10 Dryophthoridae species: *Diocalandra frumenti*,
*Trigonotarsus rugosus*, *Rhynchophorus palmarum*, *Cos-
mopolites sordidus*, *Metamasius hemipterus*, *M. callizona*,
*Sphenophorus abbreviata*, *Scyphophorus yuccae*, *Sitophi-
lus rugicollis*, and *S. granarius*. Sequences were deposited
in the GenBank database and their accession numbers are
listed later in figure 2.

Fluorescence In Situ Hybridization (FISH) Procedure

Bacteriomes were dissected in buffer A (25 mM KCl, 10 mM MgCl2, 250 mM sucrose, 35 mM Tris-HCl, pH 7.5). These tissues were homogenized in a potter and fixed in 4% paraformaldehyde solution in PBS for 30 min at 20°C. After elimination of the fixative solution, the tissues were washed by centrifugation in PBS solution (7,000 × g for 5 min). Specific oligonucleotide probes were designed by sequence alignment of 16S rDNA Dryophthoridae endosymbionts. Two probes, 5' end labeled with rhodamine were used: SOPE probe (S) 5'-TACCCCCC-TCTACGAGACTC-3' and Nardonella probe (N) 5'-ATCTAATGGCATAAGGTT-3'. Hybridization was performed at 45°C, for 4 h in a dark chamber in solution H (0.9 M NaCl, 20 mM Tris-HCl, 5 mM EDTA, 0.1% SDS, 10× Denhardt's solution) to which was added 50 ng of probe (S or N). Tissues were washed twice in the same buffer without probes at 48°C for 2 min, suspended, and mounted in Vectashield medium (Vector Laboratories) containing 4',6-diamidino-2-phenylindole (DAPI).

Phylogenetic Analyses

The alignments were performed using ClustalW
followed by manual refinement (see *Supplementary
Material*). Gaps and undetermined nucleotide sites were
removed from the data sets for all analyses. Regions where
the alignments were ambiguous were also excluded,
resulting in a data set of 1,094 nucleotide sites. Because
initial similarity analyses of the 16S rDNA sequences
performed with additional sequences from free-living
bacteria, phytopathogenic bacteria, and bacteria associated
with insect digestive tracts (accession numbers are listed
later in fig. 2) indicated that all Dryophthoridae endo-
symbionts belong to the γ-Proteobacteria group, these
sequences were used in subsequent analyses.

Phylogenetic trees were constructed using the het-
erogeneous model of DNA sequence evolution developed
by Galtier and Gouy (Galtier and Gouy 1998; Galtier,
Tourasse, and Gouy 1999), allowing for both variations of
substitution rates among sites and unequal base composi-
tions. They were then implemented in the nhml software
(URL: ftp://pbil.univ-lyon1.fr/pub/mol phylogeny/nhml).
The most likely phylogenetic tree was obtained according
to the “pruning-regrafting” method (Felsenstein 1993) by
using the shake procedure of nhml. The eval procedure
computes the log likelihood that a given tree will
correspond to the sequence data. In addition, it computes
the value of the evolutionary bias for the substitutions
between GC and AT bases for each tree branch. Such
biases quantify the tendency of an evolutionary lineage to
change the GC content of the sequence, allowing for the
estimation of (1) the equilibrium GC content of each

sequence (i.e., the GC content that a given sequence would
have if it were to evolve indefinitely with the evolutionary
bias of its terminal node) and (2) the GC contents of
ancestral sequences (i.e., sequences represented by internal
tree nodes). Such estimations and their accuracy are
detailed in Galtier and Gouy (1998).

As calculation time greatly increased with sample size
(10 to 20 days to estimate a tree with 20 sequences of
1,000 bp), methods of tree topology comparisons (Kishino
and Hasegawa 1989) as well as bootstrapping cannot be
employed with this model. A local exploration method was
therefore developed to test specific phylogenetic hypoth-
eses. This method (briefly described in the Results) is
based on an a priori fixed topology where endosymbionts
were successively added. The most likely topology is
retained according to the log-likelihood score obtained.

Phylogenetic Topology Test

The phylogenetic topology test was performed to
compare different topologies containing the same se-
quences. The alignment of 16S rDNA is bootstrapped
(500 replicates) with Seqboot software. The procedure
eval computes the log likelihood of the different topolo-
gies, to be compared, corresponding to the bootstrapping
sequence data obtained before. A *t*-test was used on log
likelihood scores obtained (500 values for each topology
tested).

Relative Rate Tests

The rates of substitution of the 16S rDNA gene
were compared between the Dryophthoridae clades and
several representatives of free-living bacteria and other
insect endosymbionts, by using the Kimura's 2-parameter
distance and covariance estimations of Li and Bousquet
(1992). Tests were performed using the RRTree software
developed by Robinson-Rechavik and Huchon (Robinson
and Huchon 2000). In each case, the *Aeromonas hay-
wardensis* sequence (AF015258) was taken as the out-
group. The null hypothesis is that the rate of substitution of
the tested clade is the same as that of the reference group
(K₁₋₀ – K₂₋₀ = 0).

Results

Endosymbiosis Occurrence in the Dryophthoridae Family

Among the 13 species analyzed histologically, 12
were shown to bear intracellular bacteria in bacteriome
structures: *Diocalandra frumenti*, *Trigonotarsus rugo-
sus*, *Yuccaborus frontalis*, *Rhynchophorus palmarum*,
*Sitophilus oryzae*, *S. zeamais*, *S. rugicollis*, *S. granarius*,
*Sphenophorus abbreviata*, *Metamasius callizona*, *Scypho-
phorus yuccae*, and *Dynamis borassi*. For *S. linearis*, no
bacteriome structures were seen. For *S. vateriae* and
*Dynamis borassi*, symbiotic status needs to be confirmed;
a bacteriome-like structure was observed in one individual
only, but no 16S sequence have been obtained successfully
(table 1).

Endosymbiotic bacteria appear pleomorphic in all
samples examined. They are rod-shaped, U-shaped, or
<figure><img src="image_2.png" /><figcaption>FIG. 1.—Histology of *Dryophthoridae* symbiosis. A. Larval bacteriome of *Trigonotarsus rugosus*. B. Endosymbiont within the cytoplasm of a bacteriocyte in *Metamasius hemipterus*. C. Spiral-shaped endosymbionts in *Sitophilus zeamais*. D. Long-shaped endosymbionts in *Sitophilus rugicollis*. FISH of *Metamasius hemipterus* endosymbiont: E. Control with DAPI fluorescence, F. Specific hybridization of the *Nardonella* probe (N). b: bacteriome; ba: bacteriocyte; g: gut. Scale bar for A, B, E, and F, 50 µm; for C and D, 10 µm.</figcaption></figure>

spiral-shaped, and their size vary from 2 to 200 µm (fig. 1). Moreover, all symbiotic species are closely associated with bacteriocytes that form bacteriomes of similar shape and location within the larvae and at the apex of the adult ovaries. The larval bacteriome is always located at the junction foregut/midgut in intimate contact with but without any communication with the intestine (Nardon et al. 2002).

To confirm that the 16S rDNA sequences generated by PCR from insect bacteriome are derived from the endosymbionts we observed, we used the FISH technique. Specific probes were designed for the R-clade (probe N) and the S-clade (Heddi et al. 1999). However, no specific probe with enough sensitivity has been obtained for the D-clade. The probe N designed from sequence alignments of R-clade endosymbionts were shown to be highly specific to bacteria contained in the bacteriome of *M. hemipterus* (fig. 1), whereas no signal could be obtained when the probe was applied to the bacteriome of *S. oryzae*. Conversely, the probe S was found to be highly specific to the S-clade endosymbionts, and no signal could be

obtained when it was applied to the bacteriome of *M. hemipterus* (Heddi et al. 1999 and data not shown).

## Phylogenetic Analyses

*Dryophthoridae Endosymbiont Analysis*

A first phylogenetic analysis was performed on the data set comprising the 13 *Dryophthoridae* endosymbiont 16S rDNA sequences. Three clades of endosymbionts were unambiguously separated regardless the phylogenetic method employed (distance, maximum likelihood, maximum parsimony) (data not shown). The D-clade includes endosymbionts of *Diocalandra frumenti* and *Trigonotarsus rugosus*; the S-clade comprises endosymbionts of *Sitophilus rugicollis*, *S. granarius*, *S. zeamais*, and *S. oryzae*; and the R-clade is represented by the endosymbionts of *Yuccaborus frontalis*, *Rhynchophorus palmarum*, *Cosmopolites sordidus*, *Sphenophorus abbreviata*, *Scyphophorus yuccae*, *Metamasius hemipterus*, and *Metamasius callizona*. These bacterial clades exhibit 53.4%, 53.8%, and 40.5% GC content, respectively, in their 16S
rDNA gene sequence. Sequences of the R-clade were highly AT biased and show many AT-rich insertions in positions 200 and 800, as described previously by Lambert and Moran (1998) in *Buchnera* endosymbionts (data not shown).

*Free-Living Bacteria Reference Topology and Local Exploration Method*

Phylogenetic analyses using standard methods (i.e., parsimony, distance, and maximum likelihood) were problematic, apparently because of extreme AT-richness and long branch attraction (Charles, Heddi, and Rahbé 2001). Therefore, a heterogeneous model of DNA evolution was used (Galtier and Gouy 1998; Galtier, Tourasse, and Gouy 1999). To reduce the space of the tree topology explored, a free-living bacteria topology was fixed a priori. The Enterobacteriaceae topology, published by Ahmad, Weisburg, and Jensen (1990) and based on fusion of genes implicated in aromatic amino acid biosynthesis, was taken as a reference. Three enteroclusters were fixed; enterocluster E1 includes the genera *Citrobacter*, *Klebsiella*, *Enterobacter*, *Escherichia*, and *Salmonella*; E2 includes the genera *Serratia* and *Erwinia*; and E3 comprises the genera *Kluyvera*, *Edwarsiella*, *Yersinia*, *Hafnia*, *Providencia*, and *Proteus*. The internal structures of the three enteroclusters were refined by “pruning regrafting” (Felsenstein 1993). To test the phylogenetic hypotheses concerning endosymbiont origin, a given endosymbiont sequence (or group of sequences) was fixed on each of branch of the reference topology; then for each new topology, including the new sequence, the corresponding log likelihood was estimated. The maximum log likelihood position was retained. Sensitivity tests entailed replacement of most of the pathogenic bacteria by other commensal free-living bacteria sequences, but no significantly different topologies were observed.

*Are the Dryophthoridae Endosymbionts Monophyletic or Polyphyletic?*

To test the hypothesis of endosymbiont monophyly, the three permutations of the three endosymbiont clades (i.e., [D, S],R; [D, R],S; and [S, R], D) were considered, and likelihoods were calculated for their placements on each of the 32 positions on the reference tree. A maximal log likelihood value of –7,389 was obtained for the (D, R), S grouping, placed at position 8 (branch numbers are reported in fig. 2). The (S, R), D grouping obtained a log likelihood value of –7,391 at the same position.

To test the hypothesis of polyphyly, the three clades were independently moved on the reference topology tree. The D-clade was unambiguously positioned on internode 5 (on *Haemophilus/Pasteurella*; fig. 2). It is noteworthy that the R-clade and the S-clade were always sister groups. These two clades were located on internode 8, with a maximal log likelihood value of –7,342. The ratio of log likelihood scores obtained (–7,342 versus –7,389) and the significant difference observed between these two topologies (phylogenetic topology test, monophyly versus

polyphyly: *P* < 10⁻⁴) supports the hypothesis of polyphyletic endosymbiont origin.

*Relative Position of Insect Endosymbionts*

Secondary endosymbionts of *Glossina pallidipes* (*Sodalis*), and endosymbionts of *Candidatus camponotii* and *Schizaphis graminum* (*Buchnera*) were added to the phylogenetic analysis on the tree topology including the three Dryophthoridae endosymbiont clades fixed as determined above. Then, local “pruning regrafting” procedures were performed to obtain the final topology (fig. 2).

*Relative and Absolute Rates of Evolution*

16S rDNA gene sequences of the R-clade exhibit much higher rates of substitution when compared to both Enterobacteriaceae and the S- and D-clades (table 2). *Buchnera* and Psyllids endosymbiont 16S rDNA sequences have previously been shown to evolve relatively faster when compared to the sequences of the free-living bacteria (Moran 1996; Fukatsu and Nikoh 1998). Here we show that endosymbiont sequences of the R-clade of the Dryophthoridae evolve even more rapidly. The relative substitution rates from the D-clade and the S-clade sequences were not found to be significantly different when compared to the Enterobacteriaceae sequences.

Using the Dryophthoridae family age estimation established by O'Meara and Farrell (personal communication), we have calculated the absolute rates of substitution (for the 16S rDNA gene) for two of the three Dryophthoridae clades. For the R-clade, which is about 100 Myr old, the estimate based on the Galtier and Gouy (1998) model of DNA evolution is 0.128 substitutions/site per 100 Myr, when considering the greatest divergences observed within the clade. Likewise, a value of 0.119 substitutions/site per 100 Myr was obtained for the S-clade (25 Myr). Therefore, the substitution rates of both the R-clade and the S-clade are much higher than those calculated for *Buchnera* (0.058 substitutions/site per 100 Myr), as well as for the enteric bacteria (0.007 to 0.018 substitutions/site per 100 Myr), (Clark, Moran, and Baumann 1999).

*Discussion*

Histological and molecular analyses reveal widespread symbiosis within the Dryophthoridae weevils. All but one Dryophthoridae species (*S. linearis*) analyzed so far were found to harbor pleomorphic intracellular bacteria in the adult and the larval bacteriomes. Bacteriomes across a wide array of weevil species are very similar in shape and location. They are always connected to the foregut/mid-gut junction in the larvae, and at the apex of the ovaries. Such uniformity of structure of the widespread symbiosis in the Dryophthoridae is consistent with long association.

Three clades of Dryphthoridae endosymbionts (R, S, and D) were defined by phylogenetic analysis, indicating that all extant Dryophthoridae endosymbionts are derived from distinct bacterial species established in three ancestral
<figure><img src="image_3.png" /><figcaption>FIG. 2.—Phylogenetic tree of *Dryophthoridae* endosymbionts. Bacterial phylogeny: maximum likelihood phylogeny based on the 16S rDNA gene sequence of Dryophthoridae and others insect endosymbionts within the γ-Proteobacteria. Numbers on the tree (mentionned in *Results*) represent the most likely positions for endosymbiont clades branching on the reference tree topology. E1, E2, and E3: Enterocluster 1, 2, and 3 defined by Ahmad, Weisburg, and Jensen (1990). P-endosymbiont: principal endosymbiont (majority and vertically transmitted), S-endosymbiont: secondary endosymbiont (minority and can be horizontally transmitted). Insect cladogram: simplify cladogram from Dryophthoridae phylogenic study from O'Meara and Farrell (personal communication).</figcaption></figure>

Dryophthoridae species. However, a close phylogenetic
proximity was observed between these three endosymbiont
clades, particularly between R and S, which are also
closely related to the other insect endosymbiotic bacteria.
This finding is consistent with the “stem clade” hypothesis
that suggests the existence within the γ3-Proteobacteria of
a limited group of bacteria that have generated intracellular

<figure><img src="image_4.png" /><figcaption>Insect cladogram</figcaption></figure>

D-clade

53.4% GC

R-clade

40.5% GC

S-clade

53.8% GC

symbiosis in many insect species and at different
evolutionary times (Charles, Heddi, and Rahbé 2001).

The R-clade endosymbionts were found in six of nine
Dryophthoridae genera, and especially within *Yuccaborus
frontalis*, the most ancestral insect of our data set
(O'Meara and Farrell, personal communication). This
clade shows much higher relative rates of substitution
when compared to both the Enterobacteriaceae and the other insect endosymbionts. A high AT bias and AT-rich insertions were also observed in the R-clade endosymbionts (40.5% GC on the 16S rDNA sequences). Estimation of the ancestral GC content of the R-clade (55.3% GC, node 8 in fig. 2) highlights this process of AT base accumulation during symbiotic evolution. Conversely, S-clade and D-clade endosymbionts are restricted to one and two genera, respectively. Moreover, they do not show a very great AT bias or a significant rates of evolution. These findings, along with dating estimations, the congruence between the host phylogeny (O'Meara and Farrell, personal communication), and the endosymbiont R-clade repartition within Dryophthoridae species, suggest that the R-clade endosymbiont represents the oldest and the ancestral bacterial infection.

The endosymbionts of the S-clade and the D-clade have been established more recently, probably by endosymbiont displacement. A hypothesis of endosymbiont replacement was postulated in aphids by Moran and Baumann (1994). More recently, a potential interplay between old-primary and recent-secondary endosymbionts was suggested by several workers who have noted a beneficial role of secondary endosymbionts for the host fitness in some aphid species (Sabater et al. 2001; Chen et al. 2000) or glossine species (Montllor, Maxmen, and Purcell 2002).

In the Dryophthoridae, the recent S-clade association is concomitant with the shift from stem-feeding, the ancestral habit in these weevils, to seed-feeding in *Sitophilus*. Likewise, the absence of intracellular bacteria in the unique asymbiotic species *S. linearis* could be interpreted as an endosymbiont loss associated with shift from monocots, the ancestral association in *Sitophilus*, to dicots. *S. linearis* feed on tamarind seeds (Fabaceae) that are much more nutritionally balanced than the cereal seeds used by the other symbiotic *Sitophilus* species (Delobel and Grenier 1993).

The D-clade is closely related to free-living parasitic bacteria such as *Haemophilus* and *Pasteurella*, which suggests that D-clade endosymbionts may also have evolved from an ancestor with a parasitic intracellular life-style. Moreover, recent studies on *Sodalis glossinidius*, the secondary endosymbiont of the tsetse flies (Dale et al. 2001), and *Sitophilus zeamais* endosymbionts (Dale et al. 2002) demonstrate that attachment and invasion of the host by the endosymbiont rely on functional genes of the type III secretion system, which are closely related to those of the pathogenic bacteria *Salmonella* and *Shigella*. Secretion systems are required for protein pathogen export and toxin delivery to the host cells. Hence, these results suggest that *Sodalis* spp. and the S-clade endosymbionts share functional infection faculties.

The position of *Sodalis glossinidius* is on the internode between *S. rugicollis* and the other *Sitophilus* species, suggesting that a horizontal transfer has occurred between *Sitophilus* weevils and tsetse fly. However, the widely separated areas of origin inferred from fossil studies for tsetse flies (Africa) and grain weevils (India) suggest, rather, an indirect transfer via some bacteria vectors.

In the early stages of symbiosis, horizontal transfers

<table><caption>**Table 2**
Evolutionary Rates of the 16S rDNA Gene Sequence in the Three Dryophthoridae Clades Relative to Other Clades Belonging to the γ-Proteobacteria and with *Aeromonas* (0) as Outgroup</caption><thead><tr><th>Tested Clade (1)</th><th>Reference Group (2)</th><th>K<sub>1-0</sub></th><th>K<sub>2-0</sub></th><th>P Value<sup>a</sup></th></tr></thead><tbody><tr><td>D-clade</td><td><i>Pasteurella/Haemophilus</i></td><td>0.117</td><td>0.140</td><td>0.01</td></tr><tr><td>D-clade</td><td>Enterobacteriaceae</td><td>0.116</td><td>0.100</td><td>0.20</td></tr><tr><td>D-clade</td><td>S-clade</td><td>0.116</td><td>0.108</td><td>0.45</td></tr><tr><td>S-clade</td><td>Enterobacteriaceae</td><td>0.112</td><td>0.106</td><td>0.29</td></tr><tr><td>S-clade</td><td><i>Glossina pallidipes</i><br>(S-endosymbiont)</td><td>0.109</td><td>0.101</td><td>0.06</td></tr><tr><td>R-clade</td><td>Enterobacteriaceae</td><td>0.233</td><td>0.102</td><td>10<sup>-7</sup></td></tr><tr><td>R-clade</td><td>S-clade</td><td>0.233</td><td>0.106</td><td>10<sup>-7</sup></td></tr><tr><td>R-clade</td><td>D-clade</td><td>0.234</td><td>0.117</td><td>10<sup>-7</sup></td></tr><tr><td>R-clade</td><td><i>Schizaphis graminum</i><br>(P-endosymbiont)</td><td>0.234</td><td>0.142</td><td>10<sup>-7</sup></td></tr><tr><td>R-clade</td><td><i>Camponotus floridanus</i><br>(Endosymbiont)</td><td>0.227</td><td>0.135</td><td>10<sup>-7</sup></td></tr></tbody></table>

of endosymbionts with pathogenic capacities could have occurred much more frequently by taking advantage of similar diets, or prey-predator and even mutualistic relationships between their respective hosts. The high homology of 16S rDNA sequences found by Aksoy, Chen, and Hypsa (1997) within the group of *Sodalis* (about 0.6% divergence) suggests recent transfers between different *Glossina* hosts. Conversely, a high divergence is observed between S-clade endosymbionts (about 6% divergence), suggesting vertical endosymbiont transmission in this group, a finding which is testable with further sampling of *Sitophilus* species.

A molecular clock calculated for the weevil hosts (O'Meara and Farrell, personal communication) allows us to observe that the S-clade endosymbionts evolve even faster than *Buchnera* (Moran 1996). Values of 0.119 and 0.058 substitutions/site per 100 Myr were measured on the S-clade endosymbionts and *Buchnera*, respectively. The difference between the relative rate test (not significant for the S-clade; table 2) and the absolute rate of substitution indicates a pronounced rate change occurring during the evolution that led from free-living bacteria to endosymbiosis establishment.

The Dryophthoridae symbiosis is unique in that an apparently single origin of the bacterial endosymbiosis in these insects is associated with three independent origins of their primary endosymbionts. Symbiosis was apparently established 100 MYA in the R-clade, followed by at least two additional symbiont substitutions, resulting from endosymbiont competition, that generated the S-clade and the D-clade, possibly in conjunction with ecological shifts in these insects.

Modeling studies indicate that parasitism by the bacteria is strongly favored when bacterial transmission occurs horizontally, with many symbionts competing among themselves and when the host defense is weak (Ewald 1995; Frank 1996). Selection pressure may eliminate host insects that are neither able to tolerate nor able to circumvent bacterial virulence. Selected hosts may possess specific strategies to control the bacteria space by inducing specific cell differentiation, the bacteriocyte

<footnote><sup>a</sup> Corresponds to the probability that Ho: $K_{1-0} = K_{2-0}$.</footnote>
for example. The high bacterial evolution rates may help to silence bacterial virulent genes through either gene mutations or deletions, thereby relaxing the selection pressure on the host and leading to insect diversification.

### Proposition of "Candidatus" Nardonella for the R-Clade Endosymbionts

Symbiotic bacteria of the R-clade are unique to the weevil family Dryophthoridae. Following Murray and Stackebrandt (1995), microorganisms partially characterized and not cultivated on laboratory media might be given the designation "Candidatus." Consequently, we propose to name the lineage corresponding to the R-clade of Dryophthoridae endosymbionts as *Candidatus* Nardonella, in honor of Professor Paul Nardon, who has first characterized endosymbionts in *Metamasius* and *Cosmopolites* (Nardon et al. 1985).

The description of "Candidatus Nardonella" is as follows: phylogenetic position, γ3-subclass of Proteobacteria; cultivation, not cultivated on cell-free media; Gram reaction, negative; morphology, pleomorphic (rod shape, U-shape, spiral shape), from 3-4 to 200 µm in length, 1-2 µm in diameter, surrounded by a mucopolysaccharide-like substance; basis of assignment, 16S rDNA sequences; association and host, intracellular symbionts of the Dryophthoridae (described in *Yuccaborus frontalis*, *Rhynchophorus palmarum*, *Cosmopolites sordidus*, *Metamasius hemipterus*, *Metamasius callizona*, *Sphenophorus abbreviata*, *Scyphophorus yuccae*); mesophilic, authors, Lefevre et al. (this study).

## Supplementary Material

Sequence Alignment of the Dryophthoridae endosymbionts is provided as Supplementary Material online (fasta format) at the journal's Web site.

### Acknowledgments

We gratefully acknowledge the colleagues who sent us the insect specimens: G. Couturier (Brazil, Belem), A. Delobel (Africa, Senegal), G. Matthew (India, Mangalore), R. Oberprieler (Australia, Queensland), Pr. Pajni (India, Chandigarh), H. Frank (USA, Florida), and, C. Nardon. We also thank P. Nardon and A. Lambert for the histological section preparations, S. Chaudier for sequencing of *S. granarius* endosymbionts, N. Galtier and M. Gouy for their critical help on the phylogenetic method, and A. Moya and M. Fernandez for critical reading. This work was supported by the French Society of Entomology (legs Germaine Cousin), Brian O'Meara kindly shared a preprint of his results on weevil phylogeny and timing of divergence.

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David Rand, Associate Editor

Accepted November 25, 2003