Diversification of Wolbachia Endosymbiont in the Culex pipiens Mosquito

Diversification of Wolbachia Endosymbiont in the Culexpipiens Mosquito

Celestine M Atyame1 Frederic Delsuc1 Nicole Pasteur1 Mylene Weill1 and Olivier Duron1

1Institut des Sciences de lrsquoEvolution CNRS UMR5554 Universite Montpellier 2 Place Eugene Bataillon Montpellier France

Corresponding author E-mail olivierduronuniv-montp2fr

Associate editor Jennifer Wernegreen

Abstract

The a-proteobacteria Wolbachia are among the most common intracellular bacteria and have recently emerged asimportant drivers of arthropod biology Wolbachia commonly act as reproductive parasites in arthropods by inducingcytoplasmic incompatibility (CI) a type of conditional sterility between hosts harboring incompatible infections In thisstudy we examined the evolutionary histories of Wolbachia infections known as wPip in the common house mosquitoCulex pipiens which exhibits the greatest variation in CI crossing patterns observed in any insect We first investigateda panel of 20 wPip strains for their genetic diversity through a multilocus scheme combining 13 Wolbachia genes BecauseWolbachia depend primarily on maternal transmission for spreading within arthropod populations we also studiedthe variability in the coinherited Cx pipiens mitochondria In total we identified 14 wPip haplotypes which all sharea monophyletic origin and clearly cluster into five distinct wPip groups The diversity of Cx pipiens mitochondria wasextremely reduced which is likely a consequence of cytoplasmic hitchhiking driven by a unique and recent Wolbachiainvasion Phylogenetic evidence indicates that wPip infections and mitochondrial DNA have codiverged through stablecotransmission within the cytoplasm and shows that a rapid diversification of wPip has occurred The observed patterndemonstrates that a considerable degree of Wolbachia diversity can evolve within a single host species over shortevolutionary periods In addition multiple signatures of recombination were found in most wPip genomic regions leadingus to conclude that the mosaic nature of wPip genomes may play a key role in their evolution

Key words endosymbiosis Wolbachia mitochondrial hitchhiking

IntroductionArthropods are commonly infected by maternally trans-mitted endosymbionts Although some endosymbiontsconfer direct benefits to their hosts by providing anabolicfunctions or resistance to pathogens (Haine 2008 Moranet al 2008) others are associated with alterations of hostreproduction (Werren et al 2008 Engelstadter and Hurst2009) These symbionts represent reproductive parasitesthat include diverse unrelated bacteria among whichthe alpha-proteobacteria Wolbachia are the most wide-spread (Duron et al 2008 Hilgenboecker et al 2008) Insome host species the successful spread of Wolbachia isachieved by biasing the hostrsquos sex ratio toward the produc-tion of females (the transmitting sex) through the induc-tion of parthenogenesis feminization or male-killing Morecommonly Wolbachia are able to induce a form of condi-tional sterility termed cytoplasmic incompatibility (CI) be-tween infected males and uninfected females or femalesinfected by incompatible strains (Werren et al 2008Engelstadter and Hurst 2009) Such manipulations enableWolbachia to spread through arthropod populations andmay drive arthropod evolution through their effects onhost phenotypes (Moran et al 2008 Werren et al 2008Engelstadter and Hurst 2009)

The dynamics of Wolbachia infections within thecommon house mosquito Culex pipiens complex remainpoorly understood The most common members of the

complex are the subspecies Cx p quinquefasciatus (Say)and Cx p pipiens (L) representing the southern and north-ern mosquito populations which are ubiquitous in tropicaland temperate regions respectively (Barr 1982) Membersof the Cx pipiens complex exhibit the greatest variation ofCI crossing types observed in arthropods thus far (Laven1967 OrsquoNeill and Paterson 1992 Guillemaud et al 1997Duron et al 2006 Atyame et al 2011) However an earlygenotyping approach using the ftsZ gene failed to revealany polymorphism between incompatible Wolbachiastrains (Guillemaud et al 1997) Further analyses used se-quences of published complete Wolbachia genomes tocharacterize polymorphic molecular markers Genomesof Wolbachia strains infecting arthropod are scattered withmobile genetic elements (MGEs) such as prophages andtransposable elements which can represent more than20 of genome content (Wu et al 2004 Klasson et al2008 2009 Salzberg et al 2009) Additionally they containan unusual high number of genes encoding proteins withankyrin (ANK) motifs which possibly mediate specific pro-teinndashprotein interactions (Sinkins et al 2005 Duron et al2007 Walker et al 2007) We further developed genotypingapproaches using ANK and MGE markers and used them toidentify more than 100 genetically distinct Wolbachiastrains (referred to as wPip strains) in natural populationsof Cx pipiens (Duron et al 2005 2006 2007 forthcomingAtyame et al 2011)

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Mol Biol Evol 28(10)2761ndash2772 2011 doi101093molbevmsr083 Advance Access publication April 22 2011 2761

Research

article

In this study we characterized the evolutionary historyof Wolbachia infections in the Cx pipiens complex byexamining the association between the wPip strains andCx pipiens mitochondrial DNA (mtDNA) variation Thepredominant mode of Wolbachia transmission withina species is vertical via the egg cytoplasm (Werren et al2008 Engelstadter and Hurst 2009) Because Wolbachiaand mitochondrial genomes are cotransmitted and there-fore in linkage disequilibrium (LD) the spread of Wolba-chia will strongly affects a hostrsquos mtDNA diversitythrough indirect selection (review in Hurst and Jiggins2005) However exceptions to strict vertical transmissionhave been found in some cases Wolbachia are also trans-ferred through horizontal transmission both within andamong different host species although the mechanismsof transfer are not well understood (Ahrens and Shoemaker2005 Baldo et al 2008 Raychoudhury et al 2009)Consequently the wide distribution of Wolbachia amongarthropods is generally assumed to result from complexinteractions between vertical and horizontal modes oftransmission modulated by their capacity to alter hostreproduction

Here we analyzed wPip variability and the associatedmtDNA diversity in 20 Cx pipiens lines encompassingunidirectionally and bidirectionally incompatible strainsthat originated from different geographic areas Our resultsshowed that the wPip strains form a monophyletic clade ofclosely related bacteria and that Cx pipiens harbors a lowlevel of mitochondrial variability which is a probable con-sequence of a recent Wolbachia invasion through cytoplas-mic hitchhiking Investigation of wPip sequences revealedextensive recombination between wPip strains althoughmultiple infections within a single mosquito were neverdetected using our markers However a congruence be-tween wPip and mtDNA phylogenies was shown demon-strating that Wolbachia mainly use maternal inheritance tospread through Cx pipiens populations The evolutionary

implications of horizontal transfers and the question ofwhether the Cx pipiensndashWolbachia association is a uniquecase or a representative example is discussed

Materials and Methods

Mosquito CollectionTwenty Cx pipiens lines from a broad geographical rangewere examined (table 1) This collection encompassed thetwo main Cx pipiens subspecies Cx p pipiens and Cxp quinquefasciatus which are naturally infected with com-patible and incompatible wPip strains (for more details seeDuron et al 2006 2007) The study also included the twolines for which the wPip genome has been sequencedwPip(Pel) (GenBank AM999887 Klasson et al 2008) andwPip(JHB) (ABZA01000000 Salzberg et al 2009)

Wolbachia MarkersThe wPip strains were first genotyped for the five house-keeping genes developed for the Wolbachia multilocusstrain typing (MLST) methodology gatB coxA hcpA ftsZand fbpA (Baldo et al 2006) and the Wolbachia surfaceprotein gene wsp (Braig et al 1998) The MLST system isclassically used to characterize the eight supergroups(AndashI) currently recognized within the Wolbachia genus(Lo et al 2007 Ros et al 2009) The polymorphism of sevenadditional genes was also examined the DNA mismatchrepair protein gene MutL (one copy in the wPip(Pel) ge-nome) 3 ANK genes ank2 (one copy) pk1 (3 identical cop-ies) and pk2 (2 identical copies) and 3 phage genes themethylase gene GP12 (4 identical copies) the putative se-creted protein gene GP15 (also known as VrlC one copy)and the regulatory protein gene RepA (one copy) None ofthese genes was amplified from Wolbachia-free Cx pipienslines which confirmed their Wolbachia origin A total of 13Wolbachia genes encompassing 19 distinct loci with a widedistribution along the wPip(Pel) chromosome were

Table 1 Description of Culex pipiens Lines and wPip Strains

Mosquito Line Abbreviation Wolbachia Strain Culex pipiens Subspecies Origin Year of Collection References

Pel Pel wPip(Pel) quinquefasciatus Sri Lanka 1984 Klasson et al (2008)JHB JHB wPip(JHB) quinquefasciatus South Africa 2001 Salzberg et al (2009)Manille-A Ma-A wPip(Ma-A) quinquefasciatus Philippines 2003 Duron et al (2006)Manille-B Ma-B wPip(Ma-B) quinquefasciatus Philippines 2003 Duron et al (2006)Kara-C Ka-C wPip(Ka-C) quinquefasciatus China 2003 Duron et al (2006)MaClo Mc wPip(Mc) quinquefasciatus California 1984 Duron et al (2005)Slab Sl wPip(Sl) quinquefasciatus California 1950 Duron et al (2005)Cotonou-A Cot-A wPip(Cot-A) quinquefasciatus Benin 2005 This studyCotonou-B Cot-B wPip(Cot-B) quinquefasciatus Benin 2005 This studyAustralie Au wPip(Au) Hybrid (quinquefasciatuspipiens) Australia 2004 Duron et al (2006)El Palmar-A Ep-A wPip(Ep-A) pipiens Spain 2005 Duron et al (2007)El Palmar-B Ep-B wPip(Ep-B) pipiens Spain 2005 Duron et al (2007)LaVar Lv wPip(Lv) pipiens France 2003 Duron et al (2005)Bifa-A Bf-A wPip(Bf-A) pipiens France 2002 Duron et al (2006)Bifa-B Bf-B wPip(Bf-B) pipiens France 2002 Duron et al (2006)Kol Ko wPip(Ko) pipiens Crete 2002 Duron et al (2006)Keo-A Ke-A wPip(Ke-A) pipiens Cyprus 2003 Duron et al (2006)Keo-B Ke-B wPip(Ke-B) pipiens Cyprus 2003 Duron et al (2006)Tunis Tn wPip(Tn) pipiens Tunisia 1992 Duron et al (2005)Istanbul Is wPip(Is) pipiens Turkey 2003 Duron et al (2005)

Atyame et al middot doi101093molbevmsr083 MBE

2762

examined (fig 1 supplementary table S1 SupplementaryMaterial online)

Culex pipiens Mitochondrial MarkersThe complete mitochondrial genome of the Pel line (15587bp) was obtained through Blast searches of the database ofwPip(Pel) contig DNA sequences from the Wellcome Trust-Sanger Institute Web site (httpwwwsangeracukProjectsW_pipientis) using the mitochondrial genome ofAedes al-bopictus (GenBank AY072044) as a probe A similar approachto obtain the mitochondrial sequences of the JHB line fromthe VectorBase Web site (httpwwwvectorbaseorg)showed several divergent mitochondrial sequences someof which exhibited low-read coverage making the JHBsequences unreliable for further analysis

A set of primers (supplementary table S2 Supplemen-tary Material online) was designed from the Pel sequencesand further used to obtain the complete mitochondrialgenomes (with the exception of the A thorn T-rich region)of additional Cx pipiens lines Specific primers were alsoused to amplify a 613-bp fragment from the NADH dehy-drogenase subunit 2 (ND2) gene a 1132-bp fragment fromthe NADH dehydrogenase subunit 5 (ND5) gene and an852-bp fragment from the cytochrome b (cytb) gene fromall the investigated Cx pipiens lines (supplementary tableS2 Supplementary Material online)

PCR Amplification and SequencingDNA was extracted from individual mosquitoes usinga cetyltrimethylammonium bromide (CTAB) protocol(Rogers and Bendich 1988) Amplification conditions were3 min at 94 C followed by 30 cycles of 94 C for 30 s 52 Cfor 30 s (58 C for MutL) and 72 C for 1ndash15 min depend-ing on the fragment size Amplified fragments were run inagarose gel (15) electrophoresis The QIAquick gel extrac-tion kit (QIAGEN Valencia CA) was then used to purify thepolymerase chain reaction (PCR) products Sequences wereobtained directly for purified products using an ABI Prism3130 sequencer with the BigDye Terminator Kit (AppliedBiosystems) The sequences have been deposited in theGenBank database (accession numbers in supplementarytables S1 and S2 Supplementary Material online)

Sequence AnalysesSequence alignments were carried out using ClustalW(Thompson et al 2002) and corrected using MEGA(Tamura et al 2007) The GBLOCKS program (Castresana2000) with default parameters was used to remove poorlyaligned positions and to obtain nonambiguous sequencealignments The number of variable sites nucleotide diver-sity (p) G thorn C content and the ratios of nonsynonymousversus synonymous substitutions (KaKs) were computedusing DNASP (Librado and Rozas 2009) Nonrandom asso-ciations between each pair of loci were estimated throughthe measure of allele LD using the D statistic (Lewontin1964 Hedrick 1987) The exact test procedure imple-mented in GENEPOP (Raymond and Rousset 1995) wasfurther used to test LD significance Statistical analysesfor intragenic recombination were performed with theSawyerrsquos test implemented in GENECONV (Sawyer1999) A Bonferroni adjustment correction for multipletesting was applied (Hochberg 1988)

Annotation of the Cx pipiens mitochondrial genomewas based on alignments with mitochondrial sequencesfrom Ae albopictus (AY072044) Ae aegypti (EU352212)Anopheles gambiae (L20934) An funestus (DQ146364)and An quadrimaculatus (L04272)

Tree ReconstructionPhylogenetic relationships were evaluated for Wolbachiaand Cx pipiens mitochondrial sequences The best-fittingmodels of sequence evolution for each data set were de-termined using the Akaike information criterion withModeltest v37 (Posada and Crandall 1998) The selectedmodel was the general time reversible model with gammadistributed among site rate variation (GTR thorn G) for bothWolbachia and mitochondrial sequence data sets Bayesianinferences (BIs) were used to reconstruct phylogenies usingMrBayes v 312 (Ronquist and Huelsenbeck 2003) Two in-dependent replicates of four Metropolis-coupled MonteCarlo Markov chains were run for 2000000 generationswith Model parameters and trees sampled every 200 gen-erations Bayesian posterior probabilities were obtainedfrom the 50 majority-rule consensus of the sampled treesafter discarding the initial burn-in period The resultingphylogenetic trees were visualized and edited in MEGA(Tamura et al 2007)

Wolbachiagenes were also analyzed within a phylogeneticnetwork framework to account for potentially conflictingsignals due to recombination (Fitch 1997) A phylogeneticnetwork was constructed based on uncorrected P distancesusing the Neighbor-net method (Bryant and Moulton 2004)implemented in SPLITSTREE (Huson and Bryant 2006)Neighbor-net is a distance-based method to construct a net-work as a generalization of all possible phylogenetictrees thatcan be reconstructed from conflicting signals in the data

Assessing the Maximum Age of MitochondrialSweepWe used the mitochondria data to infer the maximum ageof the Wolbachia infection in Cx pipiens following the

FIG 1 Map of the wPip(Pel) genome showing the position of thethirteen genes examined Number in brackets (1ndash4) indicatesidentical copies of a given gene located in different positions alongthe chromosome Black boxes indicate the locations of prophageregions the MLST and wsp genes are reported in gray

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2763

method of Rich et al (1998) This method assumes thatselection only occurs at the protein level and that DNApolymorphism in degenerate sites is neutral We used 4-fold and 2-fold synonymous sites from protein-codingmtDNA sequences to assess the age of the sweep Thenumber of 2-fold and 4-fold synonymous sites in each cod-ing region was computed with MEGA (Tamura et al 2007)and a conservative JukesndashCantor correction was applied formultiple hits

Results

Monophyletic Origin of the wPip StrainsThe MLST and wsp genes did not exhibit sequence varia-tion between the wPip strains (eight strains were examinedhere ie wPip(Sl) wPip(Tn) wPip(Ko) wPip(Lv) wPip(Is)wPip(Mc) wPip(Pel) and wPip(JHB)) establishing thatthese strains are very closely related The wPip MLSTsequence data were compared with sequences from 18other strains belonging to five distinct Wolbachia super-groups (A B D F and H) The phylogenetic tree obtainedfrom the 2079-bp concatenated MLST genes revealed thatthe wPip strains form a robust monophyletic clade withinthe B supergroup which is closely related to the wBol1strain present in the butterfly Hypolimnas bolina (identity999 supplementary fig S1 Supplementary Materialonline)

High Variability of wPip GenomesSeven of the examined Wolbachia genes were polymorphicamong the wPip genomes the DNA mismatch proteingene MutL 3 ANK genes ank2 pk1 and pk2 and 3 phagegenes GP12 GP15 and RepA Analyses revealed consider-able allelic variability among the 20 wPip strains with

2ndash8 alleles being found per gene (supplementary tableS3 Supplementary Material online) This polymorphismwas mainly due to nucleotide substitutions insertions ordeletions (indels) note that an insertion of the Tr1 trans-poson (also known as ISWpi1 see Duron et al 2005Cordaux 2008) was observed within the RepA sequenceof four wPip strains An additional source of variabilityarose from a GP15 deletion in the wPip(JHB) genome A let-ter was attributed to each distinct allele of the seven genesthe combination of which identified 14 wPip haplotypesamong the 20 strains (table 2)

Although the prophage-related genes pk1 pk2 andGP12 were found to be present in several copies in thewPip(Pel) genome divergent copies were never amplifiedfrom our wPip strains direct sequences of PCR productswere easily readable and showed no overlapping peaks Thisindicates that the different copies (if any) present in eachwPip strain examined here are identical as observed inwPip(Pel) It further shows that only mono-wPip-infections(or multiinfections by closely related wPip strains) arepresent within Cx pipiens individuals

High Recombination in wPip GenomesRecombination both within and between Wolbachiagenes can blur molecular signals and result in misleadingobservations related to strain relationships For this reasonwe checked the possibility of recombination among theseven polymorphic markers obtained here (MutL ank2pk1 pk2 GP12 GP15 and RepA) Pairwise tests for inter-genic recombination revealed significant LD for MutLank2 pk1 GP12 and GP15 (supplementary table S4 Sup-plementary Material online) Alleles at these five loci arenot randomly associated and are stably cotransmittedwithin the wPip chromosome However nonsignificantLD was found between pk2 and RepA and between thesetwo genes and the other five genes showing that recom-bination has disrupted genome clonality by shuffling theRepA and pk2 alleles among wPip strains Intragenic recom-bination was also detected for at least five genes bySawyerrsquos test (MutL pk1 pk2 GP12 and GP15 supplemen-tary table S3 Supplementary Material online) Intragenicrecombination results in identical nucleotides or aminoacid motifs in wPip strains divergent at other loci whichare readily apparent through the examination of sequencealignments (supplementary fig S2 Supplementary Materialonline)

Gene Rearrangements in wPip GenomesGenome organization of wPip strains was analyzed by com-paring the locations of the 13 genes surveyed in this studyin the wPip(Pel) chromosome and in the five majorwPip(JHB) contigs presently available (supplementaryfig S3 Supplementary Material online) There are severalrearrangements distinguishing these genomes in whichdiverse genes have been inverted (eg gatB coxA) trans-located (MutL RepA) duplicated or deleted (three andone pk1 copies are found in wPip(Pel) and wPip(JHB) re-spectively) Notably rearrangements are not limited to

Table 2 Allelic Profiles of the Seven Polymorphic wPip Genes in20 wPip Strains

Strain Gene Haplotype

MutL ank2 pk1 pk2 GP12 GP15 RepA

wPip(Pel) a a a a a a a AwPip(Cot-A) a a a a a a awPip(Cot-B) a a a a a a awPip(Ko) a a a a a a awPip(Tn) a a a a a a awPip(Ma-B) a a a a a a awPip(JHB) a a a a a - a BwPip(Ep-A) a a a d a a a CwPip(Ep-B) a a a d a a awPip(Bf-A) a a a a a a b DwPip(Lv) b e c a b b a EwPip(Au) d e c a b f a FwPip(Ke-A) c e c a d e a GwPip(Ke-B) c e c a e e a HwPip(Sl) e b b b b c b IwPip(Bf-B) e b b c b c b JwPip(Mc) b b b b b c b KwPip(Is) c c d a c d a LwPip(Ka-C) f d e a f g a MwPip(Ma-A) f d e a g g a N

NOTEmdashLetters AndashN represent the 14 wPip haplotypes Dash indicates a genedeletion

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2764

phage regions which are prone to movements within andbetween genomes but also affect housekeeping genes

Inference of wPip Strain RelationshipsPhylogenetic analyses of the 20 wPip strains using the sixwPip genes MutL ank2 pk1 pk2 GP12 and GP15 revealedsignificant topological incongruence as expected for a dataset affected by recombination (supplementary fig S4 Sup-plementary Material online) For instance the wPip(Sl)wPip(Bf-B) and wPip(Mc) strains are genetically similarfor four markers (ank2 pk1 GP12 and GP15) but appeardistantly related for two others markers (MutL and pk2)

To assess wPip strain relationships we performed phy-logenetic analyses based on the concatenated sequences ofthe seven genes The concatenated tree deduced from BIsplits the wPip clade into five groups (designated wPip-I towPip-V fig 2) However recombination can create artificialgrouping of wPip strains and network analysis was thusconducted to visualize recombination effects which wereillustrated by multiple boxes (fig 3) The evolutionary his-tory of wPip strains appears as a complex network withmultiple pathways interconnecting strains emphasizingthe mosaic nature of wPip genomes Interestingly despite

recombination the network analysis was congruent withthe Bayesian tree in recovering the same five wPip groupswith strong bootstrap values

A spatial structuring of wPip diversity emerged when thegeographic distribution of wPip groups was examined de-spite the limited number (19) of strains The most commongroup wPip-I is distributed widely from Asia to Europe(fig 4) and all wPip strains recently identified at La Reunionisland (Indian Ocean) by Atyame et al (2011) belong tothat group The wPip-V group is only found in East Asiaand the wPip-II and wPip-III groups have an apparently dis-continuous distribution with strains being found in verydistant geographic areas (eg the wPip-II strains are fromAustralia and Europe)

Low Mitochondrial Diversity in Cx pipiensCulex pipiens Pel mtDNA exhibits classical features found inother mosquito species that have been analyzed It con-tains tightly packed genes with high A thorn T content(782) There are 22 genes coding tRNAs 2 coding ribo-somal RNAs 13 genes coding subunits of enzymes involvedin oxidative phosphorylation and finally an A thorn T-richnoncoding region (supplementary fig S5 and table S5

FIG 2 Phylogenic tree of wPip strains obtained from concatenated data set (MutL ank2 pk1 pk2 GP12 GP15 and RepA sequences) byBayesian analysis Posterior probabilities obtained are shown at major nodes The scale bar is in units of substitutionssite

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Supplementary Material online) The genes are arrangedalong the chromosome in a manner similar to that of othermosquito species (Beard et al 1993 Mitchell et al 1993Krzywinski et al 1997)

The complete Cx pipiens mitochondrial genome (14856bp without the A thorn T-rich region) was sequenced from thelines Ko Tn Sl and Is and compared with the Pel genomeOverall the five mtDNA sequences displayed a very lowvariability with only 36 variable nucleotidic positions beingfound (ca 02) and two sequences were strictly identical

(lines Ko and Tn) Among the 13 protein-coding genes fivegenes (atp8 atp6 ND3 ND4L and ND6) showed nopolymorphism whereas ND2 ND5 and cytb were the mostpolymorphic (supplementary fig S5 SupplementaryMaterial online)

A likely explanation of the low mtDNA diversity in Cxpipiens populations is that cytoplasmic hitchhiking hasoccurred during Wolbachia invasion as suggested earlierby Guillemaud et al (1997) and Rasgon et al (2006) Toconfirm this hypothesis we compared the nucleotide

FIG 3 Network analysis obtained from concatenated data set (MutL ank2 pk1 pk2 GP12 GP15 and RepA sequences) using the Neighbor-netmethod Each edge (or a set of parallel edges) corresponds to a split in the data set and has length equal to the weight of the split Incompatiblesplits produced by recombination are represented by boxes in the network Only bootstrap values for major grouping are indicated The fivewPip groups (highlighted) are connected by multiple pathways resulting from recombination between Wolbachia genomes

FIG 4 Distribution of wPip haplotypes and wPip groups in Culex pipiens populations Letters and symbols represent the wPip haplotypes andwPip groups respectively wPip haplotypes recently described by Atyame et al (2011)

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diversity per site (p) at four mitochondrial loci in the 11Culicidae species for which the presence or absence of Wol-bachia has been documented (table 3 and supplementarymaterials) Only two species Cx pipiens and Ae albopictusare known to be infected whereas Wolbachia infection wasnever found in the nine other species These two Wolba-chia-infected species harbor significantly lower mtDNA di-versity than the uninfected species (Wilcoxon test W5 10P5 0008) For instance the worldwide mtDNA diversity ofCx pipiens is lower than the diversity observed in the NorthAmerican populations of Cx tarsalis an uninfected species(Venkatesan et al 2007) The low diversity of mtDNA ob-served in the Cx pipiens and Ae albopictus populations ledus to conclude that Wolbachia is most likely the causativeagent of mitochondrial sweeps in these taxa

Recent Mitochondrial Sweep in Cx pipiens ComplexWe then assessed the date of the mitochondrial sweep us-ing the nucleotide divergence of 13 protein-coding mtDNAgenes from the Cx pipiens Is line and Ae albopictus (Gen-Bank AY072044) We estimated the substitution rate forthese genes at 2-fold and 4-fold degenerate sites with a con-servative JukesndashCantor correction The genera Culex andAedes diverged approximately 172 to 226 Ma (Reidenbachet al 2009) Using the most recent estimate (172 My) themtDNA substitution rates (substitutionsiteyear) were es-timated at 51 108 and 19 108 for the 2-fold and 4-fold degenerate sites respectively whereas when using theoldest estimate (226 My) the substitution rates were 39 109 and 15 108 Among the mitochondrial genomes offive Cx pipiens lines (Is Sl Tn Ko and Pel) we observed 13and 10 nucleotide differences among the 2-fold (n5 2938)and 4-fold (n 5 1343) synonymous sites respectivelyThus this dates the Cx pipiens mitochondrial sweep be-tween 12000 and 16000 years before present (95 confi-dence interval if Culex and Aedes diverged 172 Ma) orbetween 16000 and 21000 (95 confidence interval ifthe two genera diverged 226 Ma) It is possible thatthe date of the mitochondrial sweep is even more recentas it was assumed here that the substitution rates are

constant an assumption known to overestimate diver-gence times (Ho et al 2005)

Clear Codivergence of wPip and Cx pipiensMitochondriaThe codivergence of mitochondria and wPip was assessedby studying the sequences of the three polymorphic Cxpipiens mtDNA genes (ND2 ND5 and cytb) encompassing2549 bp (164 of the whole mitochondrial genome)

Analysis of the Cx pipiens mtDNA sequences among the19 lines indicated the presence of 14 haplotypes (namedpi1 to pi14) which differed overall at 22 variable nucleotidesites (supplementary table S6 Supplementary Materialonline) The mtDNA of the Cx pipiens lines differed by only1 to 9 nucleotides confirming their very high homology(996ndash999) Phylogenetic analyses revealed two mainmitochondrial lineages (pi1 to pi5 and pi6 to pi14) withstrong branching support (fig 5A)

The concatenated mtDNA phylogeny and the wPipphylogeny were congruent (fig 5A and B) A significant as-sociation was found between mtDNA haplotypes and wPiphaplotypes (Fisherrsquos exact test P 5 3 105) as well aswPip groups (P 5 8 104) This demonstrates that wPipinfections and mtDNA have codiverged through stable co-transmission within the cytoplasm in Cx pipiens popula-tions Hence the two main mitochondrial lineagesparallel the wPip divergence pattern and strongly confirmthe wPip phylogeny Additionally Cx pipiens subspecies arenot significantly associated with wPip haplotypes (P 5

037) wPip groups (P 5 026) or mtDNA haplotypes(P 5 010) Thus Cx pipiens nuclear genomes have notcodiverged with mitochondria and wPip infections andexhibit a different evolutionary history

DiscussionHere we examined 20 isolates of Wolbachia and their as-sociated mitochondria within the Cx pipiens complex Thecombined use of Wolbachia and host mtDNA multilocussequencing revealed the processes driving the evolution ofWolbachia infections in this mosquito and raised the

Table 3 Nucleotide Diversity (p) of Mitochondrial Genes in 11 Mosquito Species (Culicidae)

Taxon (subfamily species)

p (number of sequences)Wolbachia Infection (references)

cytb ND4 COI COII

CulicinaeCulex pipiens 00021 (19) 00004 (14) 00000 (24) 00007 (8) Yes (Hertig and Wolbach 1924)Cx tarsalis mdash 00116 (64) mdash mdash No (Rasgon and Scott 2004)Culex sp mdash 00090 (10) mdash mdash No (Rasgon et al 2006)Aedes aegypti 00094 (16) 00202 (46) mdash mdash No (Kittayapong et al 2000)Aedes albopictus 00043 (14) mdash 00039 (23) mdash Yes (OrsquoNeill et al 1992)Ae caspius mdash mdash 00094 (7) 00063 (21) No (Ricci et al 2002)Ae vexans mdash mdash 00185 (7) 00084 (7) No (Kittayapong et al 2000 Ricci et al 2002)

AnophelinaeAnopheles aconitus mdash mdash 00053 (13) 00066 (35) No (Kittayapong et al 2000)An funestus 00066 (11) mdash mdash mdash No (Ricci et al 2002)Anopheles gambiae mdash mdash 00053 (48) mdash No (Ricci et al 2002)An maculipennis mdash mdash 00063 (62) mdash No (Ricci et al 2002)

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question of their likeliness to occur in other Wolbachiandasharthropod associations

Wolbachia and Mitochondrial Markers Reveala Recent Diversification of wPip StrainsThe examined MLST and wsp markers showed that wPipinfections form a robust monophyletic clade within the Bgroup of Wolbachia confirming the results of Baldo et al(2006) Although these markers are widely used to charac-terize the genetic diversity of Wolbachia even within a hostspecies (eg Dedeine et al 2004 Baldo et al 2006 2008Raychoudhury et al 2009) they displayed no variationamong the wPip infections in Cx pipiens which showsthat wPip strains have a unique and recent evolutionaryorigin As observed in other Wolbachia-infected species(Hurst and Jiggins 2005) mitochondrial diversity was lowin populations of Cx pipiens (and highly significantly lowerthan in nonndashWolbachia-infected mosquitoes) suggestingthat Wolbachia have affected mitochondrial polymor-phism in this species through cytoplasmic hitchhikingThe observed polymorphism of mitochondrial protein-coding genes indicates that the mitochondrial sweepdue to the spread of Wolbachia occurred within the last21000 years This dating is within the range of valuesclassically estimated for other Wolbachia host species ofapproximately 100000 years (Jiggins 2003 Keller et al2004 Duplouy et al 2010) and the evolutionary pathway

of wPip in Cx pipiens could be similar to other Wolbachiaarthropod associations

Multilocus typing using seven wPip polymorphicmarkers including domains of the MGE and ANK genesallowed the identification of 14 distinct wPip haplotypeswhich cluster into five distinct wPip groups This typingapproach also established that the two published wPip ge-nomes wPip(Pel) and wPip(JHB) are genetically very closeto each other compared with strains belonging to otherwPip groups in spite of their genomic differences (Salzberget al 2009) The variability of the investigated mitochon-drial markers corroborates the inferences made from thewPip markers thus in Cx pipiens different mitochondrialhaplotypes may indicate that wPip infections are differentOverall the observed genetic diversity indicates that afterthe spread of Wolbachia diversification of wPip andCx pipiens mitochondria occurred

The diversity found for wPip exhibits geographic varia-tions A remarkable degree of diversity was found in theMediterranean area where four of the five wPip groupsare found whereas a reduced diversity was observed inother regions The most common group wPip-I has a widedistribution (Asia Africa and Europe) and was also recentlyreported at La Reunion Island (Indian Ocean) (Atyameet al 2011) In contrast some wPip groups have a discon-tinuous distribution as exemplified by the wPip-II strainswhich were found in Europe and in Australia Such

FIG 5 Comparisons between phylogeny of Culex pipiens mitochondria and phylogeny of the wPip strains A mitochondrial phylogenyconstructed using BIs based on ND2 ND5 and cytb concatenated sequences Names on branches indicate the mtDNA haplotypes (piI topiXIV) B wPip phylogeny obtained from concatenated data set (MutL ank2 pk1 pk2 GP12 GP15 and RepA sequences) The five wPip groupsare reported The scale bar is in units of substitutionssite

Atyame et al middot doi101093molbevmsr083 MBE

2768

a geographic pattern is likely to be a consequence ofa recent worldwide expansion due to human activity(Raymond et al 1991 Fonseca et al 2004 2006) orandto selective advantages possibly including CI selectionHowever the 20 wPip infections investigated in this studyrepresent a restricted sampling occasionally from old mos-quito colonies and further investigations are required toimprove our knowledge of the spatial structure of the wPipgroups worldwide

wPip Strains Are Independent of Cx pipiensSubspeciesStrict vertical transmission must have favored the codiver-gence of wPip and mtDNA within shared cytoplasm How-ever there was no clear association between Cx pipienssubspecies (nuclear diversity) and cytoplasmic diversity(ie Wolbachia and mtDNA) identical wPip strains andidentical mitochondrial haplotypes were found in thetwo subspecies Cx p pipiens and Cx p quinquefasciatusA likely explanation for this is that the transfer of cytoplasmbetween Cx pipiens subspecies occurred through hybridiza-tion events as observed in Drosophila species (Rousset andSolignac 1995 Ballard 2000) and in butterfly species (Jiggins2003 Narita et al 2006 Charlat et al 2009) InCx pipiens thishypothesis is well supported by the many reports of geneticintrogression between the two subspecies in areas wherethey come into contact (Cornel et al 2003 Fonseca et al2004) Hence we can predict that DNA bar coding programsusing mtDNA will fail to discriminate between Cx p pipiensand Cx p quinquefasciatus Overall these observations sup-port the call of Hurst and Jiggins (2005) to not use mtDNAalone as a reliable means of taxa resolution

Intense Recombination Impacts the Structure ofwPip GenomesThe existence of extensive recombination among wPipstrains sheds light on the mechanisms shaping the evolu-tion of wPip genomes since recombination can influencethe adaptive dynamics of Wolbachia by creating new allelesand thus allow the emergence of new phenotypes Recom-bination between distant Wolbachia genomes has beenpreviously documented (Jiggins et al 2001 Bordensteinand Wernegreen 2004 Baldo et al 2005 Gavotte et al2007) although in this study we found recombinationamong very closely related Wolbachia genomes Evidenceof recombination was found at almost all the examinedwPip loci WO-phage genes as well as nonrelated phageloci This shows that a high level of gene flow occurs amongthe Wolbachia genomes in Cx pipiens Hence the wPipstrains do not form a set of clones in which evolution isindependent but rather represent a large population ofbacteria exchanging genetic information through lateraltransfers Although no instances of multiple infectionwere detected using our markers we must assume thatthey occur at least during a period long enough to allowrecombination between strains

Another consequence of recombination is that it canlead to misinterpretation of phylogenetic relationships

between strains However despite the extensive recombi-nation observed the wPip and mitochondrial phylogeniesare congruent recombinations have not disrupted ourgrasp of the evolutionary history of wPip strains probablybecause the contribution of recombinant regions in thephylogeny is weak compared with the diversity existingin nonrecombinant DNA fragments Therefore as sug-gested by Baldo et al (2006) the use of a multilocus ap-proach rather than single-locus analysis is requiredfor a correct understanding of the evolutionary historyof Wolbachia infections

The Cx pipiensndashWolbachia Association a UniqueCaseThe high number of wPip strains which is still certainlyunderestimated makes the Cx pipiens system remarkablebecause lower diversity is usually reported in Wolbachiaof other host species (eg Vavre et al 1999 Mercot andCharlat 2004 Charlat et al 2006 Arthofer et al 2009) How-ever it is possible that genetic variations of Wolbachia inother host species could have been missed due to themethodology generally used to characterize these bacteriaas it is generally assumed that a single Wolbachia strain ispresent within a host species when the MLST or wspmarkers are not variable In D melanogaster a single strainwMel was presumed to be present until Riegler et al (2005)identified five distinct genotypes by examining transposoninsertion sites and chromosomal inversions More recentstudies have reported different Wolbachia haplotypessolely on the basis of WO-phage genes in various host spe-cies including crickets beetles and butterflies (Charlatet al 2009 Chafee et al 2010) Hence the classical MLSTsystem is well suited to characterize Wolbachia belongingto distinct clades but specific species-typing systems basedon markers with rapid sequence evolution need to be de-veloped to investigate the Wolbachia diversity that prob-ably exists in most associations

Finally the question remains of whether the Cx pipiensndashWolbachia association is unique in term of its extremelylarge CI diversity (eg Laven 1967 OrsquoNeill and Paterson1992 Guillemaud et al 1997 Duron et al 2006) We haveclearly demonstrated that the diversity of crossing types inthis species is independent of nuclear backgrounds and re-lies solely on wPip variability (Duron et al 2006 Atyameet al 2011) The reason that a similar CI system has notbeen reported in other Wolbachia-infected species remainsa matter for speculation but it is possible that the crossingstudies conducted in Cx pipiens have been more exhaus-tive than in any other species because of the intensiveinvestigations that have been carried out for clarifyingits systematics and studying the inheritance of morpholog-ical characters since the 1930s (eg Marshall and Staley1937 Roubaud 1941 Laven 1958 1967 Rozeboom 1958Barr 1975 Narang and Seawright 1982 Irving-Bell 1983)As a result the high variability of CI crossing types wasinvestigated much earlier than the causative agent wasidentified by Yen and Barr (1971) Comparatively little workon the variability of the effects of Wolbachia infection has

Diversification of Wolbachia Endosymbiont middot doi101093molbevmsr083 MBE

2769

been conducted in most arthropods except in Drosophilaspecies such as D simulans where five distinct crossingtypes associated with distinct Wolbachia infections havethus far been identified (for review see Mercot and Charlat2004) Therefore the possibility of the existence of variablereproductive phenotypes in other host species remains tobe examined

In conclusion the use of multilocus typing combiningWolbachia and mitochondrial markers highlights the pro-cesses underlying the evolutionary dynamics of wPip infec-tions The diversification inside the wPip clade shows thata considerable amount of Wolbachia diversity can be gen-erated within a single host species in a short period of timeFurther investigations should examine the roles of recom-bination and MGE in the adaptive capacities of WolbachiaIn particular this could explain rapid changes of interac-tions between Wolbachia and their hosts (Weeks et al2007 Echaubard et al 2010) and play a key role in the evo-lution of phenotypes induced by Wolbachia Finally an im-portant question now is to determine whether the CxpipiensndashWolbachia association is a unique case or rathera representative example

Supplementary MaterialSupplementary figures S1ndashS5 and tables S1ndashS6 are availableat Molecular Biology and Evolution online (httpwwwmbe oxfordjournalsorg)

AcknowledgmentsWe are grateful to Emmanuel Douzery for his advice for anal-yses Jennifer Bernard Arnaud Berthomieu PatrickMakoundou and Sandra Unal for technical assistanceand two anonymous referees for their valuable commentsto the manuscript We acknowledge financial support fromthe PIR lsquolsquoMaladies Infectieuses et Environnementrsquorsquo fromthe CNRS All sequence data were obtained on the Envi-ronmental Genomic Platform of the IFR Montpellier-Environnement-Biodiversite This is contribution 2011043of the Institut des Sciences de lrsquoEvolution de Montpellier(UMR 5554 CNRSmdashUniversite Montpellier 2)

ReferencesAhrens ME Shoemaker D 2005 Evolutionary history of Wolbachia

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Atyame C Duron O Tortosa P Pasteur N Fort P Weill M 2011Multiple Wolbachia determinants control the evolution ofcytoplasmic incompatibilities in Culex pipiens mosquito pop-ulations Mol Ecol 20286ndash298

Baldo L Ayoub NA Hayashi CY Russell JA Stahlhut JK Werren JH2008 Insight into the routes of Wolbachia invasion high levelsof horizontal transfer in the spider genus Agelenopsis revealed byWolbachia strain and mitochondrial DNA diversity Mol Ecol17557ndash569

Baldo L Dunning Hotopp JC Jolley KA Bordenstein SR Biber SAChoudhury RR Hayashi C Maiden MC Tettelin H Werren JH

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Baldo L Lo N Werren JH 2005 Mosaic nature of the Wolbachiasurface protein J Bacteriol 1875406ndash5418

Ballard JW 2000 When one is not enough introgression ofmitochondrial DNA in Drosophila Mol Biol Evol 171126ndash1130

Barr AR 1975 Culex In King RC editor Handbook of genetics NewYork Plenum Publishing Corporation p 347ndash375

Barr AR 1982 The Culex pipiens complex In Steiner WWMTabachnik WJ Rai KS Narang S editors Recent developments inthe genetics of insect disease vectors Champaign (IL) StipesPublishing Company p 551ndash572

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Braig HR Zhou W Dobson SL OrsquoNeill SL 1998 Cloning andcharacterization of a gene encoding the major surface protein ofthe bacterial endosymbiont Wolbachia pipientis J Bacteriol1802373ndash2378

Bryant D Moulton V 2004 Neighbor-net an agglomerative methodfor the construction of phylogenetic networks Mol Biol Evol21255ndash265

Castresana J 2000 Selection of conserved blocks from multiplealignments for their use in phylogenetic analysis Mol Biol Evol17540ndash552

Chafee ME Funk DJ Harrison RG Bordenstein SR 2010Lateral phage transfer in obligate intracellular bacteria (Wolbachia)verification from natural populations Mol Biol Evol 27501ndash505

Charlat S Duplouy A Hornett EA Dyson EA Davies N Roderick GKWedell N Hurst GD 2009 The joint evolutionary histories ofWolbachia and mitochondria in Hypolimnas bolina BMC EvolBiol 964

Charlat S Engelstadter J Dyson EA Hornett EA Duplouy ATortosa P Davies N Roderick GK Wedell N Hurst GD 2006Competing selfish genetic elements in the butterfly Hypolimnasbolina Curr Biol 162453ndash2458

Cordaux R 2008 ISWpi1 from Wolbachia pipientis defines a novelgroup of insertion sequences within the IS5 family Gene40920ndash27

Cornel AJ McAbee RD Rasgon J Stanich MA Scott TW Coetzee M2003 Differences in extent of genetic introgression betweensympatric Culex pipiens and Culex quinquefasciatus (DipteraCulicidae) in California and South Africa J Med Entomol4036ndash51

Dedeine F Vavre F Shoemaker DD Bouletreau M 2004 Intra-individual coexistence of a Wolbachia strain required for hostoogenesis with two strains inducing cytoplasmic incompatibilityin the wasp Asobara tabida Evolution 582167ndash2174

Duplouy A Hurst GD OrsquoNeill SL Charlat S 2010 Rapid spread ofmale-killing Wolbachia in the butterfly Hypolimnas bolina J EvolBiol 23231ndash235

Duron O Bernard C Unal S Berthomieu A Berticat C Weill M2006 Tracking factors modulating cytoplasmic incompatibilitiesin the mosquito Culex pipiens Mol Ecol 153061ndash3071

Duron O Bouchon D Boutin S Bellamy L Zhou L Engelstadter JHurst GD 2008 The diversity of reproductive parasites amongarthropods Wolbachia do not walk alone BMC Biol 627

Duron O Boureux A Echaubard P Berthomieu A Berticat C Fort PWeill M 2007 Variability and expression of ankyrin domaingenes in Wolbachia variants infecting the mosquito Culexpipiens J Bacteriol 1894442ndash4448

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Duron O Lagnel J Raymond M Bourtzis K Fort P Weill M 2005Transposable element polymorphism of Wolbachia in themosquito Culex pipiens evidence of genetic diversity superin-fection and recombination Mol Ecol 141561ndash1573

Duron O Raymond M Weill M Forthcoming Many compatibleWolbachia strains coexist within natural populations of Culexpipiens mosquito Heredity

Echaubard P Duron O Agnew P Sidobre C Noel V Weill MMichalakis Y 2010 Rapid evolution of Wolbachia density ininsecticide resistant Culex pipiens Heredity 10415ndash19

Engelstadter J Hurst GDD 2009 The ecology and evolution ofmicrobes that manipulate host reproduction Annu Rev Ecol EvolSyst 40127ndash149

Fitch WM 1997 Networks and viral evolution J Mol Evol 44(Suppl1)S65ndashS75

Fonseca DM Keyghobadi N Malcolm CA Mehmet C Schaffner FMogi M Fleischer RC Wilkerson RC 2004 Emerging vectors inthe Culex pipiens complex Science 3031535ndash1538

Fonseca DM Smith JL Wilkerson RC Fleischer RC 2006 Pathwaysof expansion and multiple introductions illustrated by largegenetic differentiation among worldwide populations of thesouthern house mosquito Am J Trop Med Hyg 74284ndash289

Gavotte L Henri H Stouthamer R Charif D Charlat S Bouletreau MVavre F 2007 A survey of the bacteriophage WO in theendosymbiotic bacteria Wolbachia Mol Biol Evol 24427ndash435

Guillemaud T Pasteur N Rousset F 1997 Contrasting levels ofvariability between cytoplasmic genomes and incompatibilitytypes in the mosquito Culex pipiens Proc Biol Sci 264245ndash251

Haine ER 2008 Symbiont-mediated protection Proc Biol Sci275353ndash361

Hedrick PW 1987 Gametic disequilibrium measures proceed withcaution Genetics 117331ndash341

Hertig M Wolbach SB 1924 Studies on Rickettsia-like micro-organisms in insects J Med Res 44329ndash3747

Hilgenboecker K Hammerstein P Schlattmann P Telschow AWerren JH 2008 How many species are infected withWolbachiamdasha statistical analysis of current data FEMS Micro-biol Lett 281215ndash220

Ho SYW Phillips MJ Cooper A Drummond AJ 2005 Timedependency of molecular rate estimates and systematic over-estimation of recent divergence times Mol Biol Evol 221561ndash1568

Hochberg Y 1988 A sharper Bonferroni procedure for multiple testsof significance Biometrika 75800ndash802

Hurst GDD Jiggins FM 2005 Problems with mitochondrial DNA asa marker in population phylogeographic and phylogeneticstudies the effects of inherited symbionts Proc Biol Sci2721525ndash1534

Huson DH Bryant D 2006 Application of phylogenetic networks inevolutionary studies Mol Biol Evol 23254ndash267

Irving-Bell RJ 1983 Cytoplasmic incompatibility within and betweenCulex molestus and Cx quinquefasciatus (Diptera Culicidae) JMed Entomol 2044ndash48

Jiggins FM 2003 Male-killing Wolbachia and mitochondrial DNAselective sweeps hybrid introgression and parasite populationdynamics Genetics 1645ndash12

Jiggins FM von Der Schulenburg JH Hurst GD Majerus ME 2001Recombination confounds interpretations of Wolbachia evolu-tion Proc Biol Sci 2681423ndash1427

Keller GP Windsor DM Saucedo JM Werren JH 2004 Reproductiveeffects and geographical distributions of two Wolbachia strainsinfecting the Neotropical beetle Chelymorpha alternans Boh(Chrysomelidae Cassidinae) Mol Ecol 132405ndash2420

Kittayapong P Baisley KJ Baimai V OrsquoNeill SL 2000 Distributionand diversity of Wolbachia infections in Southeast Asianmosquitoes (Diptera Culicidae) J Med Entomol 37340ndash345

Klasson L Walker T Sebaihia M et al (12 co-authors) 2008Genome evolution of Wolbachia strain wPip from the Culexpipiens group Mol Biol Evol 251877ndash1887

Klasson L Westberg J Sapountzis P et al (12 co-authors) 2009 Themosaic genome structure of the Wolbachia wRi strain infectingDrosophila simulans Proc Natl Acad Sci U S A 1065725ndash5730

Krzywinski J Grushko OG Besansky NJ 1997 Analysis of thecomplete mitochondrial DNA from Anopheles funestus animproved dipteran mitochondrial genome annotation anda temporal dimension of mosquito evolution Mol Phyl Evol39417ndash423

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Lewontin RC 1964 The interaction of selection and linkage IGeneral considerations heterotic models Genetics 4949ndash67

Librado P Rozas J 2009 DnaSP v5 a software for comprehensiveanalysis of DNA polymorphism data Bioinformatics 251451ndash1452

Lo N Paraskevopoulos C Bourtzis K OrsquoNeill SL Werren JHBordenstein SR Bandi C 2007 Taxonomic status of theintracellular bacterium Wolbachia pipientis Int J Syst EvolMicrobiol 57654ndash657

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Mercot H Charlat S 2004 Wolbachia infections in Drosophilamelanogaster and D simulans polymorphism and levels ofcytoplasmic incompatibility Genetica 12051ndash59

Mitchell SE Cockburn AF Seawright JA 1993 The mitochondrialgenome of Anopheles quadrimaculatus species A completenucleotide sequence and gene organization Genome 361058ndash1073

Moran NA McCutcheon JP Nakabachi A 2008 Genomics andevolution of heritable bacterial symbionts Ann Rev Genet42165ndash190

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Posada D Crandall KA 1998 MODELTEST testing the model ofDNA substitution Bioinformatics 14817ndash818

Rasgon JL Cornel AJ Scott TW 2006 Evolutionary history ofa mosquito endosymbiont revealed through mitochondrialhitchhiking Proc Biol Sci 2731603ndash1611

Rasgon JL Scott TW 2004 An initial survey for Wolbachia(Rickettsiales Rickettsiaceae) infections in selected Californiamosquitoes (Diptera Culicidae) J Med Entomol 41255ndash257

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Raychoudhury R Baldo L Oliveira DC Werren JH 2009 Modes ofacquisition of Wolbachia horizontal transfer hybrid introgres-sion and codivergence in the Nasonia species complexEvolution 63165ndash183

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Raymond M Rousset F 1995 Genepop (version 12) a populationgenetics software for exact tests and ecumenicism J Hered86248ndash249

Reidenbach KR Cook S Bertone MA Harbach RE Wiegmann BMBesansky NJ 2009 Phylogenetic analysis and temporal di-versification of mosquitoes (Diptera Culicidae) based on nucleargenes and morphology BMC Evol Biol 9298

Ricci I Cancrini G Gabrielli S DrsquoAmelio S Favi G 2002 Searching forWolbachia (Rickettsiales Rickettsiaceae) in mosquitoes (DipteraCulicidae) large polymerase chain reaction survey and newidentifications J Med Entomol 39562ndash567

Rich SM Licht MC Hudson RR Ayala FJ 1998 Malariarsquos eveevidence of a recent population bottleneck throughout theworld populations of Plasmodium falciparum Proc Natl Acad SciU S A 954425ndash4430

Riegler M Sidhu M Miller WJ OrsquoNeill SL 2005 Evidence for a globalWolbachia replacement in Drosophila melanogaster Curr Biol151428ndash1433

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Ros VI Fleming VM Feil EJ Breeuwer JA 2009 How diverse is thegenus Wolbachia Multiple-gene sequencing reveals a putativelynew Wolbachia supergroup recovered from spider mites (AcariTetranychidae) Appl Environ Microbiol 751036ndash1043

Roubaud E 1941 Phenomenes drsquoamixie dans les intercroisementsde Culicides du groupe pipiens C R Acad Sci 212257ndash259

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Rozeboom LE 1958 Hybridization of Culex pipiens fatigansWiedemann from the Philippine Islands with American strains

of Culex pipiens group (Diptera Culicidae) Am J Trop Med Hyg7526ndash530

Salzberg SL Puiu D Sommer DD Nene V Lee NH 2009 Thegenome sequence of Wolbachia endosymbiont of Culexquinquefasciatus JHB J Bacteriol 1911725

Sawyer SA 1999 GENECONV a computer package for the statisticaldetection of gene conversion [Internet] Distributed by theauthor Department of Mathematics Washington University inSt Louis Missouri USA Available (2011) from httpwwwmathwustledu~sawyergeneconv

Sinkins SP Walker T Lynd AR Steven AR Makepeace BL Godfray HCParkhill J 2005 Wolbachia variability and host effects on crossingtype in Culex mosquitoes Nature 436257ndash260

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Thompson JD Gibson TJ Higgins DG 2002 Multiple sequencealignment using ClustalW and ClustalX Curr Protoc Bioinfor-matics Chapter 2Unit 23 httpwwwncbinlmnihgovpubmed18792934

Vavre F Fleury F Lepetit D Fouillet P Bouletreau M 1999Phylogenetic evidence for horizontal transmission of Wolbachiain hostndashparasitoid associations Mol Biol Evol 161711ndash1723

Venkatesan M Westbrook CJ Hauer MC Rasgon JL 2007 Evidencefor a population expansion in the West Nile Virus vector Culextarsalis Mol Biol Evol 241208ndash1218

Walker T Klasson L Sebaihia M Sanders MJ Thomson NR Parkhill JSinkins SP 2007 Ankyrin repeat domain-encoding genes in thewPip strain of Wolbachia from the Culex pipiens group BMCBiol 539

Weeks AR Turelli M Harcombe WR Reynolds KT Hoffmann AA2007 From parasite to mutualist rapid evolution of Wolbachiain natural populations of Drosophila PLoS Biol 5e114

Werren JH Baldo L Clark ME 2008 Wolbachia master manipu-lators of invertebrate biology Nat Rev Microbiol 6741ndash751

Wu M Sun LV Vamathevan J et al (30 co-authors) 2004Phylogenomics of the reproductive parasite Wolbachia pipientiswMel a streamlined genome overrun by mobile genetic elementsPLoS Biol 2E69

Yen JH Barr AR 1971 New hypothesis of the cause of cytoplasmicincompatibility in Culex pipiens L Nature 232657ndash658

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2772