Célestine Atyame Nten

Frédéric Delsuc

Nicole Pasteur

Mylène Weill

Olivier Duron
email: olivier.duron@univ-montp2.fr

Célestine M Atyame

Diversification of Wolbachia Endosymbiont in the Culex pipiens Mosquito

Keywords: Endosymbiosis, Wolbachia, mitochondrial hitchhiking

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Introduction

Arthropods are commonly infected by maternally transmitted endosymbionts. Although some endosymbionts confer direct benefits to their hosts by providing anabolic functions or resistance to pathogens [START_REF] Haine | Symbiont-mediated protection[END_REF][START_REF] Moran | Genomics and evolution of heritable bacterial symbionts[END_REF], others are associated with alterations of host reproduction [START_REF] Werren | Wolbachia: master manipulators of invertebrate biology[END_REF][START_REF] Engelstadter | The ecology and evolution of microbes that manipulate host reproduction[END_REF]. These symbionts represent reproductive parasites that include diverse unrelated bacteria, among which the alpha-proteobacteria Wolbachia are the most widespread [START_REF] Duron | The diversity of reproductive parasites among arthropods: Wolbachia do not walk alone[END_REF][START_REF] Hilgenboecker | How many species are infected with Wolbachia?--A statistical analysis of current data[END_REF]. In some host species, the successful spread of Wolbachia is achieved by biasing the host's sex ratio toward the production of females (the transmitting sex) through the induction of parthenogenesis, feminization or male-killing.

More commonly, Wolbachia are able to induce a form of conditional sterility, termed cytoplasmic incompatibility (CI), between infected males and uninfected females or females infected by incompatible strains [START_REF] Werren | Wolbachia: master manipulators of invertebrate biology[END_REF][START_REF] Engelstadter | The ecology and evolution of microbes that manipulate host reproduction[END_REF]. Such manipulations enable Wolbachia to spread through arthropod populations and may drive arthropod evolution through their effects on host phenotypes [START_REF] Moran | Genomics and evolution of heritable bacterial symbionts[END_REF][START_REF] Werren | Wolbachia: master manipulators of invertebrate biology[END_REF][START_REF] Engelstadter | The ecology and evolution of microbes that manipulate host reproduction[END_REF].

The dynamics of Wolbachia infections within the common house mosquito, Culex pipiens complex, remain poorly understood. The most common members of the complex are the subspecies Cx. p. quinquefasciatus (Say) and Cx. p. pipiens (L.), representing the southern and northern mosquito populations, which are ubiquitous in tropical and temperate regions, respectively [START_REF] Barr | The Culex pipiens complex[END_REF]. Members of the Cx. pipiens complex exhibit the greatest variation of CI crossing types observed in arthropods thus far [START_REF] Laven | Speciation and evolution in Culex pipiens in[END_REF][START_REF] O'neill | Crossing type variability associated with cytoplasmic incompatibility in Australian populations of the mosquito Culex quinquefasciatus Say[END_REF][START_REF] Guillemaud | Contrasting levels of variability between cytoplasmic genomes and incompatibility types in the mosquito Culex pipiens[END_REF]Duron et al. 2006;Atyame et al. in press). However, an early genotyping approach using the ftsZ gene failed to reveal any polymorphism between incompatible Wolbachia strains [START_REF] Guillemaud | Contrasting levels of variability between cytoplasmic genomes and incompatibility types in the mosquito Culex pipiens[END_REF]. Further analyses used sequences of published complete Wolbachia genomes to characterize polymorphic molecular markers. Genomes of Wolbachia strains infecting arthropod are scattered with mobile genetic elements (MGE), such as prophages and transposable elements, which can represent more than 20% of genome content [START_REF] Wu | Phylogenomics of the reproductive parasite Wolbachia pipientis wMel: a streamlined genome overrun by mobile genetic elements[END_REF][START_REF] Klasson | Genome evolution of Wolbachia strain wPip from the Culex pipiens group[END_REF][START_REF] Salzberg | The genome sequence of Wolbachia endosymbiont of Culex quinquefasciatus JHB[END_REF][START_REF] Klasson | The mosaic genome structure of the Wolbachia wRi strain infecting Drosophila simulans[END_REF]). Additionally, they contain an unusual high number of genes encoding proteins with ankyrin (ANK) motif(s), which possibly mediate specific proteinprotein interactions [START_REF] Sinkins | Wolbachia variability and host effects on crossing type in Culex mosquitoes[END_REF][START_REF] Duron | Variability and expression of ankyrin domain genes in Wolbachia variants infecting the mosquito Culex pipiens[END_REF][START_REF] Walker | Ankyrin repeat domain-encoding genes in the wPip strain of Wolbachia from the Culex pipiens group[END_REF]). We further developed genotyping approaches using ANK and MGE markers and used them to identify more than 100 genetically distinct Wolbachia strains (referred to as wPip strains) in natural populations of Cx. pipiens [START_REF] Duron | Transposable element polymorphism of Wolbachia in the mosquito Culex pipiens: evidence of genetic diversity, superinfection and recombination[END_REF]Duron et al. 2006;[START_REF] Duron | Variability and expression of ankyrin domain genes in Wolbachia variants infecting the mosquito Culex pipiens[END_REF]Atyame et al. in press;Duron, Raymond, and Weill in press).

In this study, we characterized the evolutionary history of Wolbachia infections in the Cx. pipiens complex by examining the association between the wPip strains and Cx. pipiens mitochondrial DNA variation. The predominant mode of Wolbachia transmission within a species is vertical, via the egg cytoplasm [START_REF] Werren | Wolbachia: master manipulators of invertebrate biology[END_REF][START_REF] Engelstadter | The ecology and evolution of microbes that manipulate host reproduction[END_REF]. Because Wolbachia and mitochondrial genomes are co-transmitted and, therefore, in linkage disequilibrium, the spread of Wolbachia will strongly affects a host's mtDNA diversity through indirect selection (review in [START_REF] Hurst | Problems with mitochondrial DNA as a marker in population, phylogeographic and phylogenetic studies: the effects of inherited symbionts[END_REF]. However, exceptions to strict vertical transmission have been found; in some cases, Wolbachia are also transferred through horizontal transmission both within and among different host species, although the mechanisms of transfer are not well understood (Ahrens and Shoemaker 2005; [START_REF] Werren | Wolbachia: master manipulators of invertebrate biology[END_REF][START_REF] Raychoudhury | Modes of acquisition of Wolbachia: horizontal transfer, hybrid introgression, and codivergence in the Nasonia species complex[END_REF]. Consequently, the wide distribution of Wolbachia among arthropods is generally assumed to result from complex interactions between vertical and horizontal modes of transmission, modulated by their capacity to alter host reproduction.

Here, we analyzed wPip variability and the associated mtDNA diversity in twenty Cx. pipiens lines encompassing uni-and bidirectionally incompatible strains that originated from different geographic areas. Our results showed that the wPip strains form a monophyletic clade of closely related bacteria and that Cx. pipiens harbors a low level of mitochondrial variability, which is a probable consequence of a recent Wolbachia invasion through cytoplasmic hitchhiking. Investigation of wPip sequences revealed extensive recombination between wPip strains, although multiple infections within a single mosquito were never detected using our markers. However, a congruence between wPip and mtDNA phylogenies was shown, demonstrating that Wolbachia mainly use maternal inheritance to spread through Cx. pipiens populations. The evolutionary implications of horizontal transfers and the question of whether the Cx. pipiens -Wolbachia association is a unique case or a representative example is discussed.

Materials and Methods

Mosquito collection

Twenty Cx. pipiens lines from a broad geographical range were examined (Table 1). This collection encompassed the two main Cx. pipiens subspecies, Cx. p. pipiens and Cx. p. quinquefasciatus, which are naturally infected with compatible and incompatible wPip strains (for more details, see Duron et al. 2006;[START_REF] Duron | Variability and expression of ankyrin domain genes in Wolbachia variants infecting the mosquito Culex pipiens[END_REF]). The study also included the two lines for which the wPip genome has been sequenced, wPip(Pel) (GenBank AM999887; [START_REF] Klasson | Genome evolution of Wolbachia strain wPip from the Culex pipiens group[END_REF] and wPip(JHB) (ABZA01000000; [START_REF] Salzberg | The genome sequence of Wolbachia endosymbiont of Culex quinquefasciatus JHB[END_REF].

Wolbachia markers

The wPip strains were first genotyped for the five housekeeping genes developed for the Wolbachia MultiLocus Strain Typing (MLST) methodology, gatB, coxA, hcpA, ftsZ and fbpA [START_REF] Baldo | Multilocus sequence typing system for the endosymbiont Wolbachia pipientis[END_REF], and the Wolbachia surface protein gene wsp [START_REF] Braig | Cloning and characterization of a gene encoding the major surface protein of the bacterial endosymbiont Wolbachia pipientis[END_REF]). The MLST system is classically used to characterize the eight supergroups (A to I) currently recognized within the Wolbachia genus [START_REF] Lo | Taxonomic status of the intracellular bacterium Wolbachia pipientis[END_REF][START_REF] Ros | How diverse is the genus Wolbachia? Multiple-gene sequencing reveals a putatively new Wolbachia supergroup recovered from spider mites (Acari: Tetranychidae)[END_REF]). The polymorphism of seven additional genes was also examined: the DNA mismatch repair protein gene MutL (one copy in the wPip(Pel) genome), three ANK genes, ank2 (one copy), pk1 (three identical copies), and pk2 (two identical copies), and three phage genes, the methylase gene GP12 (four identical copies), the putative secreted protein gene GP15 (also known as VrlC; one copy) and the regulatory protein gene RepA (one copy). None of these genes was amplified from Wolbachia-free Cx. pipiens lines, which confirmed their Wolbachia origin. A total of 13 Wolbachia genes, encompassing 19 distinct loci with a wide distribution along the wPip(Pel) chromosome, were examined (Figure 1, Table S1).

Culex pipiens mitochondrial markers

The complete mitochondrial genome of the Pel line (15,587 bp) was obtained through BLAST searches of the database of wPip(Pel) contig DNA sequences from the Wellcome Trust-Sanger Institute website (http://www.sanger.ac.uk/Projects/W_pipientis/) using the mitochondrial genome of Aedes albopictus (GenBank AY072044) as a probe. A similar approach to obtain the mitochondrial sequences of the JHB line from the VectorBase website (http://www.vectorbase.org/) showed several divergent mitochondrial sequences, some of which exhibited low read coverage, making the JHB sequences unreliable for further analysis.

A set of primers (Table S2) was designed from the Pel sequences and further used to obtain the complete mitochondrial genomes (with the exception of the A+T-rich region) of additional Cx. pipiens lines. Specific primers were also used to amplify a 613 bp fragment from the NADH dehydrogenase subunit 2 (ND2) gene, a 1,132 bp fragment from the NADH dehydrogenase subunit 5 (ND5) gene and an 852 bp fragment from the cytochrome b (cytb) gene from all the investigated Cx. pipiens lines (Table S2).

PCR amplification and sequencing

DNA was extracted from individual mosquitoes using a CetylTrimethylAmmonium Bromide (CTAB) protocol [START_REF] Rogers | Extraction of DNA from plant tissues[END_REF]. Amplification conditions were 3 min at 94°C, followed by 30 cycles of 94°C for 30 s, 52°C for 30 s (58°C for MutL), and 72°C for 1 to 1.5 min depending on the fragment size. Amplified fragments were run in agarose gel (1.5%) electrophoresis. The QIAquick gel extraction kit (QIAGEN, Valencia, CA) was then used to purify the PCR products. Sequences were obtained directly for purified products using an ABI Prism 3130 sequencer with the BigDye Terminator Kit (Applied Biosystems). The sequences have been deposited in the GenBank database (accession numbers in Tables S1 andS2).

Sequence analyses

Sequence alignments were carried out using CLUSTALW [START_REF] Thompson | Multiple sequence alignment using ClustalW and ClustalX[END_REF] and corrected using MEGA [START_REF] Tamura | MEGA4: Molecular evolutionary genetics analysis (MEGA) software version 4.0[END_REF]). The GBLOCKS program [START_REF] Castresana | Selection of conserved blocks from multiple alignments for their use in phylogenetic analysis[END_REF] with default parameters was used to remove poorly aligned positions and to obtain nonambiguous sequence alignments. The number of variable sites (VI), nucleotide diversity (π), G+C content and the ratios of nonsynonymous versus synonymous substitutions (Ka/Ks) were computed using DNASP [START_REF] Librado | DnaSP v5: A software for comprehensive analysis of DNA polymorphism data[END_REF]. Nonrandom associations between each pair of loci were estimated through the measure of allele linkage disequilibrium (LD) using the D′ statistic [START_REF] Lewontin | The interaction of selection and linkage. I. General considerations, heterotic models[END_REF][START_REF] Hedrick | Gametic disequilibrium measures: proceed with caution[END_REF]. The exact test procedure implemented in GENEPOP [START_REF] Raymond | Genepop (version 1.2), a population genetics software for exact tests and ecumenicism[END_REF] was further used to test LD significance.

Statistical analyses for intragenic recombination were performed with the Sawyer's test implemented in GENECONV [START_REF] Sawyer | GENECONV: A computer package for the statistical detection of gene conversion[END_REF]. A Bonferonni adjustment correction for multiple testing was applied [START_REF] Hochberg | A sharper Bonferroni procedure for multiple tests of significance[END_REF].

Annotation of the Cx. pipiens mitochondrial genome was based on alignments with mitochondrial sequences from Ae. albopictus (AY072044), Ae. aegypti (EU352212), Anopheles gambiae (L20934), An. funestus (DQ146364) and An. quadrimaculatus (L04272).

Tree reconstruction

Phylogenetic relationships were evaluated for Wolbachia and Cx. pipiens mitochondrial sequences. The best-fitting models of sequence evolution for each dataset were determined using the Akaike information criterion with MODELTEST v3.7 [START_REF] Posada | MODELTEST: testing the model of DNA substitution[END_REF].

The selected model was the General Time Reversible model, with Gamma distributed amongsite rate variation (GTR+G) for both Wolbachia and mitochondrial sequence datasets.

Bayesian inferences (BI) were used to reconstruct phylogenies using Mr. Bayes v 3.1.2 [START_REF] Ronquist | MrBayes 3: Bayesian phylogenetic inference under mixed models[END_REF]. Two independent replicates of four Metropolis-coupled Monte Carlo Markov chains were run for 2,000,000 generations with Model parameters and trees sampled every 200 generations. Bayesian posterior probabilities were obtained from the 50% majority-rule consensus of the sampled trees after discarding the initial burn-in period.

The resulting phylogenetic trees were visualized and edited in MEGA [START_REF] Tamura | MEGA4: Molecular evolutionary genetics analysis (MEGA) software version 4.0[END_REF].

Wolbachia genes were also analyzed within a phylogenetic network framework to account for potentially conflicting signals due to recombination [START_REF] Fitch | Networks and viral evolution[END_REF]. A phylogenetic network was constructed based on uncorrected P-distances using the Neighbor-net method [START_REF] Bryant | Neighbor-net: an agglomerative method for the construction of phylogenetic networks[END_REF] implemented in SPLITSTREE [START_REF] Huson | Application of phylogenetic networks in evolutionary studies[END_REF]. Neighbor-net is a distance-based method to construct a network as a generalization of all possible phylogenetic trees that can be reconstructed from conflicting signals in the data.

Assessing the maximum age of mitochondrial sweep

We used the mitochondria data to infer the maximum age of the Wolbachia infection in Cx.

pipiens following the method of [START_REF] Rich | Malaria's Eve: evidence of a recent population bottleneck throughout the world populations of Plasmodium falciparum[END_REF]. This method assumes that selection only occurs at the protein level and that DNA polymorphism in degenerate sites is neutral. We used fourfold and twofold synonymous sites from protein coding mtDNA sequences to assess the age of the sweep. The number of twofold and fourfold synonymous sites in each coding region was computed with MEGA [START_REF] Tamura | MEGA4: Molecular evolutionary genetics analysis (MEGA) software version 4.0[END_REF], and a conservative Jukes-Cantor correction was applied for multiple hits.

Results

Monophyletic origin of the wPip strains

The MLST and wsp genes did not exhibit sequence variation between the wPip strains (eight strains were examined here, i.e. wPip(Sl), wPip(Tn), wPip(Ko), wPip(Lv), wPip(Is), wPip (Mc), wPip(Pel) and wPip(JHB)), establishing that these strains are very closely related.

The wPip MLST sequence data were compared to sequences from 18 other strains belonging to five distinct Wolbachia supergroups (A, B, D, F and H). The phylogenetic tree obtained from the 2,079-bp concatenated MLST genes revealed that the wPip strains form a robust monophyletic clade within the B supergroup, which is closely related to the wBol1 strain present in the butterfly Hypolimnas bolina (identity > 99.9%; Figure S1).

High variability of wPip genomes

Seven of the examined Wolbachia genes were polymorphic among the wPip genomes: the DNA mismatch protein gene MutL, three ANK genes ank2, pk1 and pk2, and three phage genes GP12, GP15 and RepA. Analyses revealed considerable allelic variability among the 20 wPip strains, with 2-8 alleles being found per gene (Table S3). This polymorphism was mainly due to nucleotide substitutions, insertions or deletions (indels); note that an insertion of the Tr1 transposon (also known as ISWpi1; see [START_REF] Duron | Transposable element polymorphism of Wolbachia in the mosquito Culex pipiens: evidence of genetic diversity, superinfection and recombination[END_REF][START_REF] Cordaux | ISWpi1 from Wolbachia pipientis defines a novel group of insertion sequences within the IS5 family[END_REF]) was observed within the RepA sequence of three wPip strains. An additional source of variability arose from a GP15 deletion in the wPip(JHB) genome. A letter was attributed to each distinct allele of the seven genes, the combination of which identified 14 wPip haplotypes among the twenty strains (Table 2).

Although the prophage related genes pk1, pk2 and GP12 were found to be present in several copies in the wPip(Pel) genome, divergent copies were never amplified from our wPip strains: direct sequences of PCR products were easily readable and showed no overlapping peaks. This indicates that the different copies (if any) present in each wPip strain examined here are identical, as observed in wPip(Pel). It further shows that only mono-wPip-infections (or multiinfections by closely related wPip strains) are present within Cx. pipiens individuals.

High recombination in wPip genomes

Recombination, both within and between Wolbachia genes, can blur molecular signals and result in misleading observations related to strain relationships. For this reason, we checked the possibility of recombination among the seven polymorphic markers obtained here (MutL,ank2,pk1,pk2,GP12,GP15 and RepA). Pairwise tests for intergenic recombination revealed significant linkage disequilibrium (LD) for MutL, ank2, pk1, GP12, and GP15 (Table S4).

Alleles at these five loci are not randomly associated and are stably co-transmitted within the wPip chromosome. However, nonsignificant LD was found between pk2 and RepA and between these two genes and the other five genes, showing that recombination has disrupted genome clonality by shuffling the RepA and pk2 alleles among wPip strains. Intragenic recombination was also detected for at least five genes by Sawyer's test (MutL, pk1, pk2, GP12 and GP15; Table S3). Intragenic recombination results in identical nucleotides or amino acid motifs in wPip strains divergent at other loci, which are readily apparent through the examination of sequence alignments (Figure S2).

Gene rearrangements in wPip genomes

Genome organization of wPip strains was analyzed by comparing the locations of the thirteen genes surveyed in this study in the wPip(Pel) chromosome and in the five major wPip(JHB) contigs presently available (Figure S3). There are several rearrangements distinguishing these genomes, in which diverse genes have been inverted (e.g. gatB, coxA), translocated (MutL, RepA), duplicated or deleted (three and one pk1 copies are found in wPip(Pel) and wPip(JHB), respectively). Notably, rearrangements are not limited to phage regions, which are prone to movements within and between genomes, but also affect housekeeping genes.

Inference of wPip strain relationships

Phylogenetic analyses of the 20 wPip strains using the six wPip genes, MutL, ank2, pk1, pk2, GP12 and GP15, revealed significant topological incongruence, as expected for a dataset affected by recombination (Figure S4). For instance, the wPip(Sl), wPip(Bf-B) and wPip (Mc) strains are genetically similar for four markers (ank2, pk1, GP12 and GP15), but appear distantly related for two others markers (MutL and pk2).

To assess wPip strain relationships, we performed phylogenetic analyses based on the concatenated sequences of the seven genes. The concatenated tree deduced from Bayesian inference splits the wPip clade into five groups (designated wPip-I to wPip-V; Figure 2). However, recombination can create artificial grouping of wPip strains, and network analysis was thus conducted to visualize recombination effects, which were illustrated by multiple boxes (Figure 3). The evolutionary history of wPip strains appears as a complex network with multiple pathways interconnecting strains, emphasizing the mosaic nature of wPip genomes.

Interestingly, despite recombination, the network analysis was congruent with the Bayesian tree in recovering the same five wPip groups with strong bootstrap values.

A spatial structuring of wPip diversity emerged when the geographic distribution of wPip groups was examined, despite the limited number (19) of strains. The most common group, wPip-I, is distributed widely from Asia to Europe (Figure 4), and all wPip strains recently identified at La Réunion island (Indian Ocean) by Atyame et al. (in press) belong to that group. The wPip-V group is only found in East Asia, and the wPip-II and wPip-III groups have an apparently discontinuous distribution, with strains being found in very distant geographic areas (for example, the wPip-II strains are from Australia and Europe).

Low mitochondrial diversity in Cx. pipiens

Cx. pipiens Pel mitochondrial DNA exhibits classical features found in other mosquito species that have been analyzed. It contains tightly packed genes with high A+T content (78.2%). There are twenty-two genes coding tRNAs, two coding ribosomal RNAs, thirteen genes coding subunits of enzymes involved in oxidative phosphorylation and, finally, an A+T rich noncoding region (Figure S5 and Table S5). The genes are arranged along the chromosome in a manner similar to that of other mosquito species [START_REF] Beard | The mitochondrial genome of the mosquito Anopheles gambiae: DNA sequence, genome organization, and comparisons with mitochondrial sequences of other insects[END_REF][START_REF] Mitchell | The mitochondrial genome of Anopheles quadrimaculatus species A: complete nucleotide sequence and gene organization[END_REF][START_REF] Krzywinski | Analysis of the complete mitochondrial DNA from Anopheles funestus: An improved dipteran mitochondrial genome annotation and a temporal dimension of mosquito evolution[END_REF].

The complete Cx. pipiens mitochondrial genome (14,856 bp without the A+T rich region) was sequenced from the lines Ko, Tn, Sl and Is and compared to the Pel genome. Overall, the five mtDNA sequences displayed a very low variability, with only 36 variable nucleotidic positions being found (ca. 2‰), and two sequences were strictly identical (lines Ko and Tn).

Among the 13 protein-coding genes, five genes (atp8, atp6, ND3, ND4L and ND6) showed no polymorphism, whereas ND2, ND5 and cytb were the most polymorphic (Figure S5). A likely explanation of the low mtDNA diversity in Cx. pipiens populations is that cytoplasmic hitchhiking has occurred during Wolbachia invasion, as suggested earlier by Guillemaud et al. [START_REF] Guillemaud | Contrasting levels of variability between cytoplasmic genomes and incompatibility types in the mosquito Culex pipiens[END_REF]) and Rasgon et al. [START_REF] Rasgon | Evolutionary history of a mosquito endosymbiont revealed through mitochondrial hitchhiking[END_REF]. To confirm this hypothesis, we compared the nucleotide diversity per site (π) at four mitochondrial loci in the eleven Culicidae species for which the presence or absence of Wolbachia has been documented (Table 3 and supplementary materials). Only two species, Cx. pipiens and Ae. albopictus, are known to be infected, whereas Wolbachia infection was never found in the nine other species. These two Wolbachia-infected species harbor significantly lower mtDNA diversity than the uninfected species (Wilcoxon test, W=10, P = 0.008). For instance, the worldwide mtDNA diversity of Cx. pipiens is lower than the diversity observed in the North American populations of Cx. tarsalis, an uninfected species [START_REF] Venkatesan | Evidence for a population expansion in the West Nile Virus vector Culex tarsalis[END_REF]. The low diversity of mtDNA observed in the Cx. pipiens and Ae. albopictus populations led us to conclude that Wolbachia is most likely the causative agent of mitochondrial sweeps in these taxa.

Recent mitochondrial sweep in Cx. pipiens complex

We then assessed the date of the mitochondrial sweep using the nucleotide divergence of 13 protein-coding mtDNA genes from the Cx. pipiens Is line and Ae. albopictus (GenBank AY072044). We estimated the substitution rate for these genes at twofold and fourfold degenerate sites with a conservative Jukes-Cantor correction. The genera Culex and Aedes diverged approximately 172 to 226 My ago [START_REF] Reidenbach | Phylogenetic analysis and temporal diversification of mosquitoes (Diptera: Culicidae) based on nuclear genes and morphology[END_REF]). Using the most recent estimate (172 My), the mtDNA substitution rates (substitution/site/year) were estimated at 5.1×10

-8 and 19×10 -8 for the twofold and fourfold degenerate sites, respectively, whereas, when using the oldest estimate (226 My), the substitution rates were 39×10 -9 and 15×10 -8 .

Among the mitochondrial genomes of five Cx. pipiens lines (Is, Sl, Tn, Ko and Pel), we observed 13 and 10 nucleotide differences among the twofold (n=2,938) and fourfold (n=1,343) synonymous sites, respectively. Thus, this dates the Cx. pipiens mitochondrial sweep between 12,000 and 16,000 years before present (95% confidence interval, if Culex and Aedes diverged 172 My ago) or between 16,000 and 21,000 (95% confidence interval, if the two genera diverged 226 My ago). It is possible that the date of the mitochondrial sweep is even more recent as it was assumed here that the substitution rates are constant, an assumption known to overestimate divergence times [START_REF] Ho | Time dependency of molecular rate estimates and systematic overestimation of recent divergence times[END_REF].

Clear co-divergence of wPip and Cx. pipiens mitochondria

The co-divergence of mitochondria and wPip was assessed by studying the sequences of the three polymorphic Cx. pipiens mtDNA genes (ND2, ND5 and cytb), encompassing 2,549 bp (16.4% of the whole mitochondrial genome).

Analysis of the Cx. pipiens mtDNA sequences among the 19 lines indicated the presence of 14 haplotypes (named pi1 to pi14), which differed overall at 22 variable nucleotide sites (Table S6). The mtDNA of the Cx. pipiens lines differed by only one to nine nucleotides, confirming their very high homology (99.6-99.9%). Phylogenetic analyses revealed two main mitochondrial lineages (pi1 to pi5 and pi6 to pi14) with strong branching support (Figure 5A).

The concatenated mtDNA phylogeny and the wPip phylogeny were congruent (Figure 5A and B). A significant association was found between mtDNA haplotypes and wPip haplotypes (Fisher's exact test, P = 310 -5 ), as well as wPip groups (P = 810 -4 ). This demonstrates that wPip infections and mitochondrial DNA have co-diverged through stable co-transmission within the cytoplasm in Cx. pipiens populations. Hence, the two main mitochondrial lineages parallel the wPip divergence pattern and strongly confirm the wPip phylogeny. Additionally, Cx. pipiens subspecies are not significantly associated with wPip haplotypes (P = 0.37), wPip groups (P = 0.26) or mtDNA haplotypes (P = 0.10). Thus, Cx. pipiens nuclear genomes have not co-diverged with mitochondria and wPip infections and exhibit a different evolutionary history.

Discussion

Here, we examined 20 isolates of Wolbachia and their associated mitochondria within the Cx.

pipiens complex. The combined use of Wolbachia and host mtDNA multi-locus sequencing revealed the processes driving the evolution of Wolbachia infections in this mosquito and raised the question of their likeliness to occur in other Wolbachia-arthropod associations.

Wolbachia and mitochondrial markers reveal a recent diversification of wPip strains

The examined MLST and wsp markers showed that wPip infections form a robust monophyletic clade within the B group of Wolbachia, confirming the results of Baldo et al. [START_REF] Baldo | Multilocus sequence typing system for the endosymbiont Wolbachia pipientis[END_REF]. Although these markers are widely used to characterize the genetic diversity of Wolbachia, even within a host species (e.g. [START_REF] Dedeine | Intra-individual coexistence of a Wolbachia strain required for host oogenesis with two strains inducing cytoplasmic incompatibility in the wasp Asobara tabida[END_REF][START_REF] Baldo | Multilocus sequence typing system for the endosymbiont Wolbachia pipientis[END_REF][START_REF] Werren | Wolbachia: master manipulators of invertebrate biology[END_REF][START_REF] Raychoudhury | Modes of acquisition of Wolbachia: horizontal transfer, hybrid introgression, and codivergence in the Nasonia species complex[END_REF], they displayed no variation among the wPip infections in Cx. pipiens, which shows that wPip strains have a unique and recent evolutionary origin. As observed in other Wolbachia infected species [START_REF] Hurst | Problems with mitochondrial DNA as a marker in population, phylogeographic and phylogenetic studies: the effects of inherited symbionts[END_REF], mitochondrial diversity was low in populations of Cx. pipiens (and highly significantly lower than in non-Wolbachia-infected mosquitoes), suggesting that Wolbachia have affected mitochondrial polymorphism in this species through cytoplasmic hitchhiking. The observed polymorphism of mitochondrial protein coding genes indicates that the mitochondrial sweep due to the spread of Wolbachia occurred within the last 21,000 years. This dating is within the range of values classically estimated for other Wolbachia host species of ca. < 100,000 years, [START_REF] Jiggins | Male-killing Wolbachia and mitochondrial DNA: selective sweeps, hybrid introgression and parasite population dynamics[END_REF][START_REF] Keller | Reproductive effects and geographical distributions of two Wolbachia strains infecting the Neotropical beetle, Chelymorpha alternans Boh. (Chrysomelidae, Cassidinae)[END_REF][START_REF] Duplouy | Rapid spread of male-killing Wolbachia in the butterfly Hypolimnas bolina[END_REF], and the evolutionary pathway of wPip in Cx. pipiens could be similar to other Wolbachia/arthropod associations.

Multi-locus typing using seven wPip polymorphic markers, including domains of the MGE and ANK genes, allowed the identification of 14 distinct wPip haplotypes, which cluster into five distinct wPip groups. This typing approach also established that the two published wPip genomes, wPip(Pel) and wPip(JHB), are genetically very close to each other compared to strains belonging to other wPip groups, in spite of their genomic differences [START_REF] Salzberg | The genome sequence of Wolbachia endosymbiont of Culex quinquefasciatus JHB[END_REF]). The variability of the investigated mitochondrial markers corroborates the inferences made from the wPip markers; thus, in Cx. pipiens, different mitochondrial haplotypes may indicate that wPip infections are different. Overall, the observed genetic diversity indicates that, after the spread of Wolbachia, diversification of wPip and Cx. pipiens mitochondria occurred.

The diversity found for wPip exhibits geographic variations. A remarkable degree of diversity was found in the Mediterranean area, where four of the five wPip groups are found, whereas a reduced diversity was observed in other regions. The most common group, wPip-I, has a wide distribution (Asia, Africa and Europe) and was also recently reported at La Réunion Island (Indian Ocean) (Atyame et al. in press). In contrast, some wPip groups have a discontinuous distribution, as exemplified by the wPip-II strains, which were found in Europe and in Australia. Such a geographic pattern is likely to be a consequence of a recent worldwide expansion due to human activity [START_REF] Raymond | Worldwide migration of amplified insecticide resistance genes in mosquitoes[END_REF][START_REF] Fonseca | Emerging vectors in the Culex pipiens complex[END_REF][START_REF] Fonseca | Pathways of expansion and multiple introductions illustrated by large genetic differentiation among worldwide populations of the southern house mosquito[END_REF] or/and to selective advantages, possibly including CI selection. However, the 20 wPip infections investigated in this study represent a restricted sampling, occasionally from old mosquito colonies, and further investigations are required to improve our knowledge of the spatial structure of the wPip groups worldwide.

wPip strains are independent of Cx. pipiens subspecies Strict vertical transmission must have favored the co-divergence of wPip and mtDNA within shared cytoplasm. However, there was no clear association between Cx. pipiens subspecies (nuclear diversity) and cytoplasmic diversity (i.e. Wolbachia and mtDNA): identical wPip strains and identical mitochondrial haplotypes were found in the two subspecies, Cx. p. pipiens and Cx. p. quinquefasciatus. A likely explanation for this is that the transfer of cytoplasm between Cx. pipiens subspecies occurred through hybridization events, as observed in Drosophila species [START_REF] Rousset | Evolution of single and double Wolbachia symbioses during speciation in the Drosophila simulans complex[END_REF][START_REF] Ballard | When one is not enough: introgression of mitochondrial DNA in Drosophila[END_REF] and in butterfly species [START_REF] Jiggins | Male-killing Wolbachia and mitochondrial DNA: selective sweeps, hybrid introgression and parasite population dynamics[END_REF][START_REF] Narita | Genetic structure of sibling butterfly species affected by Wolbachia infection sweep: evolutionary and biogeographical implications[END_REF][START_REF] Charlat | The joint evolutionary histories of Wolbachia and mitochondria in Hypolimnas bolina[END_REF]). In Cx. pipiens, this hypothesis is well supported by the many reports of genetic introgression between the two subspecies in areas where they come into contact [START_REF] Cornel | Differences in extent of genetic introgression between sympatric Culex pipiens and Culex quinquefasciatus (Diptera: Culicidae) in California and South Africa[END_REF][START_REF] Fonseca | Emerging vectors in the Culex pipiens complex[END_REF]). Hence, we can predict that DNA barcoding programs using mtDNA will fail to discriminate between Cx. p. pipiens and Cx. p. quinquefasciatus. Overall, these observations support the call of Hurst and Jiggins [START_REF] Hurst | Problems with mitochondrial DNA as a marker in population, phylogeographic and phylogenetic studies: the effects of inherited symbionts[END_REF] to not use mtDNA alone as a reliable means of taxa resolution.

Intense recombination impacts the structure of wPip genomes

The existence of extensive recombination among wPip strains sheds light on the mechanisms shaping the evolution of wPip genomes since recombination can influence the adaptive dynamics of Wolbachia by creating new alleles and thus allow the emergence of new phenotypes. Recombination between distant Wolbachia genomes has been previously documented [START_REF] Jiggins | Recombination confounds interpretations of Wolbachia evolution[END_REF][START_REF] Bordenstein | Bacteriophage flux in endosymbionts (Wolbachia): infection frequency, lateral transfer, and recombination rates[END_REF][START_REF] Baldo | Mosaic nature of the Wolbachia surface protein[END_REF][START_REF] Gavotte | A Survey of the bacteriophage WO in the endosymbiotic bacteria Wolbachia[END_REF], although in this study, we found recombination among very closely related Wolbachia genomes. Evidence of recombination was found at almost all of the examined wPip loci, WO-phage genes, as well as nonrelated phage loci. This shows that a high level of gene flow occurs among the Wolbachia genomes in Cx. pipiens. Hence, the wPip strains do not form a set of clones in which evolution is independent but, rather, represent a large population of bacteria exchanging genetic information through lateral transfers.

Although no instances of multiple infection were detected using our markers, we must assume that they occur, at least during a period long enough to allow recombination between strains.

Another consequence of recombination is that it can lead to misinterpretation of phylogenetic relationships between strains. However, despite the extensive recombination observed, the wPip and mitochondrial phylogenies are congruent: recombinations have not disrupted our grasp of the evolutionary history of wPip strains, probably because the contribution of recombinant regions in the phylogeny is weak compared to the diversity existing in nonrecombinant DNA fragments. Therefore, as suggested by [START_REF] Baldo | Multilocus sequence typing system for the endosymbiont Wolbachia pipientis[END_REF], the use of a multi-locus approach, rather than single locus analysis, is required for a correct understanding of the evolutionary history of Wolbachia infections.

The Cx. pipiens-Wolbachia association, a unique case?

The high number of wPip strains, which is still certainly underestimated, makes the Cx.

pipiens system remarkable because lower diversity is usually reported in Wolbachia of other host species (e.g. [START_REF] Vavre | Phylogenetic evidence for horizontal transmission of Wolbachia in host-parasitoid associations[END_REF][START_REF] Mercot | Wolbachia infections in Drosophila melanogaster and D. simulans: polymorphism and levels of cytoplasmic incompatibility[END_REF][START_REF] Charlat | Competing selfish genetic elements in the butterfly Hypolimnas bolina[END_REF]Arthofer et al. 2009). However, it is possible that genetic variations of Wolbachia in other host species could have been missed due to the methodology generally used to characterize these bacteria, as it is generally assumed that a single Wolbachia strain is present within a host species when the MLST or wsp markers are not variable. In D. melanogaster, a single strain, wMel, was presumed to be present until Riegler et al. [START_REF] Riegler | Evidence for a global Wolbachia replacement in Drosophila melanogaster[END_REF]) identified five distinct genotypes by examining transposon insertion sites and chromosomal inversions. More recent studies have reported different Wolbachia haplotypes solely on the basis of WO phage genes in various host species, including crickets, beetles and butterflies [START_REF] Charlat | The joint evolutionary histories of Wolbachia and mitochondria in Hypolimnas bolina[END_REF][START_REF] Chafee | Lateral phage transfer in obligate intracellular bacteria (Wolbachia): verification from natural populations[END_REF]. Hence, the classical MLST system is well suited to characterize Wolbachia belonging to distinct clades, but specific species typing systems based on markers with rapid sequence evolution need to be developed to investigate the Wolbachia diversity that probably exists in most associations.

Finally, the question remains of whether the Cx. pipiens -Wolbachia association is unique in term of its extremely large CI diversity (e.g. [START_REF] Laven | Speciation and evolution in Culex pipiens in[END_REF][START_REF] O'neill | Crossing type variability associated with cytoplasmic incompatibility in Australian populations of the mosquito Culex quinquefasciatus Say[END_REF][START_REF] Guillemaud | Contrasting levels of variability between cytoplasmic genomes and incompatibility types in the mosquito Culex pipiens[END_REF]Duron et al. 2006). We have clearly demonstrated that the diversity of crossing types in this species is independent of nuclear backgrounds and 20 relies solely on wPip variability (Duron et al. 2006;Atyame et al. in press). The reason that a similar CI system has not been reported in other Wolbachia-infected species remains a matter for speculation, but it is possible that the crossing studies conducted in Cx. pipiens have been more exhaustive than in any other species because of the intensive investigations that have been carried out for clarifying its systematics and studying the inheritance of morphological characters since the 1930s (e.g. [START_REF] Marshall | Some notes regarding the morphological and biological differentiation of Culex pipiens Linnaeus and Culex molestus Forskal (Diptera, Culicidae)[END_REF][START_REF] Roubaud | Phénomènes d'amixie dans les intercroisements de Culicides du groupe pipiens[END_REF][START_REF] Laven | Speciation by cytoplasmic isolation in the Culex pipiens complex[END_REF][START_REF] Rozeboom | Hybridization of Culex pipiens fatigans Wiedemann from the Philippine Islands with American strains of Culex pipiens group ( Diptera: culicidae)[END_REF][START_REF] Laven | Speciation and evolution in Culex pipiens in[END_REF][START_REF] Barr | Handbook of Genetics[END_REF][START_REF] Narang | Linkage relationships and genetic mapping in Culex and Anopheles[END_REF][START_REF] Irving-Bell | Cytoplasmic incompatibility within and between Culex molestus and Cx. quinquefasciatus (Diptera: Culicidae)[END_REF]. As a result, the high variability of CI crossing types was investigated much earlier than the causative agent was identified by [START_REF] Yen | New hypothesis of the cause of cytoplasmic incompatibility in Culex pipiens L[END_REF] Comparatively little work on the variability of the effects of Wolbachia infection has been conducted in most arthropods, except in Drosophila species, such as D. simulans, where five distinct crossing types associated with distinct Wolbachia infections have thus far been identified (for review see [START_REF] Mercot | Wolbachia infections in Drosophila melanogaster and D. simulans: polymorphism and levels of cytoplasmic incompatibility[END_REF]. Therefore, the possibility of the existence of variable reproductive phenotypes in other host species remains to be examined.

In conclusion, the use of multi-locus typing combining Wolbachia and mitochondrial markers highlights the processes underlying the evolutionary dynamics of wPip infections. The diversification inside the wPip clade shows that a considerable amount of Wolbachia diversity can be generated within a single host species in a short period of time. Further investigations should examine the roles of recombination and MGE in the adaptive capacities of Wolbachia.

In particular, this could explain rapid changes of interactions between Wolbachia and their hosts [START_REF] Weeks | From parasite to mutualist: rapid evolution of Wolbachia in natural populations of Drosophila[END_REF][START_REF] Echaubard | Rapid evolution of Wolbachia density in insecticide resistant Culex pipiens[END_REF]) and play a key role in the evolution of phenotypes induced by Wolbachia. Finally, an important question now is to determine whether the Cx. pipiens -Wolbachia association is a unique case or, rather, a representative example. An. funestus 0.0066 (11) _ _ _ no [START_REF] Ricci | Searching for Wolbachia (Rickettsiales: Rickettsiaceae) in mosquitoes (Diptera: Culicidae): large polymerase chain reaction survey and new identifications[END_REF] An. gambiae _ _ 0.0053 (48) _ no [START_REF] Ricci | Searching for Wolbachia (Rickettsiales: Rickettsiaceae) in mosquitoes (Diptera: Culicidae): large polymerase chain reaction survey and new identifications[END_REF] An. maculipennis _ _ 0.0063 (62) _ no [START_REF] Ricci | Searching for Wolbachia (Rickettsiales: Rickettsiaceae) in mosquitoes (Diptera: Culicidae): large polymerase chain reaction survey and new identifications[END_REF] Table 3. Nucleotide diversity (π) of mitochondrial genes in eleven mosquito species (Culicidae).

Figure legends

Figure1.

  Figure1. Map of the wPip(Pel) genome showing the position of the thirteen genes examined.

Figure 2 .

 2 Figure 2. Phylogenic tree of wPip strains obtained from concatenated data set (MutL, ank2,

Figure 3 .

 3 Figure 3. Network analysis obtained from concatenated data set(MutL, ank2, pk1, pk2,

Figure 4 .

 4 Figure 4. Distribution of wPip haplotypes and wPip groups in Culex pipiens populations.

  Figure 1

Table 1 .

 1 Description of Culex pipiens lines and wPip strains. 759

	Mosquito line	Abbreviation	Wolbachia strain	Culex pipiens subspecies	Origin	Year of collection Reference
	Pel	Pel	wPip(Pel)	quinquefasciatus	Sri Lanka	(Klasson et al. 2008)
	JHB	JHB	wPip(JHB)	quinquefasciatus	South Africa	(Salzberg et al. 2009)
	Manille-A	Ma-A	wPip(Ma-A)	quinquefasciatus	Philippines	(Duron et al. 2006)
	Manille-B	Ma-B	wPip(Ma-B)	quinquefasciatus	Philippines	(Duron et al. 2006)
	Kara-C	Ka-C	wPip(Ka-C)	quinquefasciatus	China	(Duron, Fort, and Weill 2006)
	MaClo	Mc	wPip(Mc)	quinquefasciatus	California	(Duron et al. 2005)
	Slab	Sl	wPip(Sl)	quinquefasciatus	California	(Duron et al. 2005)
	Cotonou-A	Cot-A	wPip(Cot-A)	quinquefasciatus	Benin	This study
	Cotonou-B	Cot-B	wPip(Cot-B)	quinquefasciatus	Benin	This study
	Australie	Au	wPip(Au)	hybrid (quinquefasciatus/pipiens) Australia	(Duron, Fort, and Weill 2006)
	El Palmar-A	Ep-A	wPip(Ep-A)	pipiens	Spain	(Duron et al. 2007)
	El Palmar-B	Ep-B	wPip(Ep-B)	pipiens	Spain	(Duron et al. 2007)
	LaVar	Lv	wPip(Lv)	pipiens	France	(Duron et al. 2005)
	Bifa-A	Bf-A	wPip(Bf-A)	pipiens	France	(Duron et al. 2006)
	Bifa-B	Bf-B	wPip(Bf-B)	pipiens	France	(Duron et al. 2006)
	Kol	Ko	wPip(Ko)	pipiens	Crete	(Duron, Fort, and Weill 2006)
	Keo-A	Ke-A	wPip(Ke-A)	pipiens	Cyprus	(Duron et al. 2006)
	Keo-B	Ke-B	wPip(Ke-B)	pipiens	Cyprus	(Duron et al. 2006)
	Tunis	Tn	wPip(Tn)	pipiens	Tunisia	(Duron et al. 2005)
	Istanbul	Is	wPip(Is)	pipiens	Turkey	(Duron et al. 2005)

Table 2 .

 2 Allelic profiles of the seven polymorphic wPip genes in 20 wPip strains. Letters A to N represent the 14 wPip haplotypes. Dash indicates a gene deletion.

	Taxon (subfamily, species)	cytb	ND4	π (no of sequences) COI	COII	Wolbachia infection (reference)
	Culicinae
	Culex pipiens	0.0021 (19)	0.0004 (14)	0.0000 (24)	0.0007 (8)	yes (Hertig and Wolbach 1924)
	Cx. tarsalis	_	0.0116 (64)	_	_	no (Rasgon and Scott 2004)
	Culex sp.	_	0.0090 (10)	_	_	no (Rasgon, Cornel, and Scott 2006)
	Aedes aegypti	0.0094 (16)	0.0202 (46)	_	_	no (Kittayapong et al. 2000)
	Ae. albopictus	0.0043 (14)	_		0.0039 (23)	_	yes (O'Neill et al. 1992)
	Ae. caspius	_	_		0.0094 (7)	0.0063 (21)	no (Ricci et al. 2002)
	Ae. vexans	_	_		0.0185 (7)	0.0084 (7)	no (Kittayapong et al. 2000; Ricci et al. 2002)
	Anophelinae
	Anopheles aconitus	_	_		0.0053 (13)	0.0066 (35)	no (Kittayapong et al. 2000)

Acknowledgments

We are grateful to Emmanuel Douzery for his advice for analyses, Jennifer Bernard, Arnaud Berthomieu, Patrick Makoundou and Sandra Unal for technical assistance, and two anonymous referees for their valuable comments to the manuscript. We acknowledge financial support from the PIR "Maladies Infectieuses et Environnement" from the CNRS. All sequence data were obtained on the Environmental Genomic Platform of the IFR Montpellier-Environnement-Biodiversité. This is contribution 2011.nx of the Institut des Sciences de l'Evolution de Montpellier (UMR 5554 CNRS -Université Montpellier 2).