BACKGROUND
symbiotic associations with the intracellular bacterium wolbachia are extremely widespread in insects
<cit> .
wolbachia is typically maternally inherited through the egg cytoplasm and has evolved a variety of interactions with its hosts, exerting subtle effects such as manipulation of host reproduction or protection against natural enemies
<cit> .
each of these effects is advantageous to infected females and thus enables wolbachia to spread rapidly through insect populations.
these effects are also of ecological and evolutionary importance to the particular host species that is infected, potentially inducing reproductive isolation or driving changes in sexuality
<cit> .
mosquitoes of the culex pipiens complex have long been recognized to exhibit a great variability of effects associated with wolbachia.
in this host, wolbachia, known as wpip, is associated with cytoplasmic incompatibility , a sperm-egg incompatibility between infected males and uninfected females, so that infected females have a reproductive advantage
<cit> .
apart from this simple case, ci has been also observed in a number of cases between males and females carrying incompatible wpip strains
<cit> .
five housekeeping genes developed for wolbachia multilocus strain typing
<cit>  and the wolbachia surface protein gene wsp present no variation among these wpip strains, showing that they delineate a recent and monophyletic clade into the b wolbachia supergroup
<cit> .
the recent examination of fast evolving markers, such as the ank <dig> and pk <dig> genes encoding proteins with ankyrin  motifs, revealed the existence of more than  <dig> genetically distinct wpip strains
<cit> , belonging to five distinct subclades and further referred as wpip-i to wpip-v groups
<cit> .
since their molecular characterization, several recent studies have further showed that some wpip strains are mutually incompatible but also that some others, although genetically distinct, are fully compatible
<cit> .
meanwhile, wpip has also emerged as a conditional mutualist that protects cx.
pipiens against mortality induced by the avian malaria parasite plasmodium relictum <cit> .
overall, these studies revealed that wpip is an important associate of most mosquitoes in the cx.
pipiens complex potentially driving its evolution.
how wpip infections interact with the genetic structure within the cx.
pipiens complex remains still unclear.
mosquitoes in this complex have a global distribution in all temperate and tropical regions, with a recent history of association with human migration
<cit> .
the complex encompasses a group of genetically closely related taxa with distinct behavioural and physiological traits that greatly influence their distribution.
the most obvious variable traits include larval habitat preference, vertebrate feeding pattern, mating behaviour, gonotrophic development and ability to enter into diapause during the winter
<cit> .
despite these differences, members of the cx.
pipiens complex have controversial taxonomic statuses  and, even though they are genetically distinct, they remain difficult to separate morphologically.
four species, or subspecies according to the authors, are frequently recognized as members of the complex: cx.
pipiens, cx.
quinquefasciatus, cx.
australicus, and cx.
globocoxitus, as well as two subspecies: cx.
pipiens pipiens in europe and north and south africa, and cx.
p. pallens, in asia.
in addition, two sympatric forms, pipiens and molestus, are also encountered in cx.
p. pipiens in the northern hemisphere.
cx.
quinquefasciatus, commonly known as the southern house mosquito, exists all across the tropics and the lower latitudes of temperate regions.
cx.
australicus and cx.
globocoxitus are restricted to australia and are poorly known.
although the exact taxonomic status of the cx.
pipiens members remains controversial, their close evolutionary association has been repeatedly supported by genetic analyses and the relative abundance of hybrids in areas where distributions overlap
<cit> .
remarkably, whilst wpip was never detected in cx.
australicus and cx.
globocoxitus, infection frequency was near or at fixation in almost all populations of both cx.
pipiens and cx.
quinquefasciatus <cit> .
when first described, any of the five wpip groups were found associated within given infected members of the cx.
pipiens complex
<cit> .
however, only a limited number of cx.
pipiens and cx.
quinquefasciatus laboratory lines have been examined, making this result difficult to interpret.
this issue is of special importance since wolbachia may either produce reproductive isolation between host populations infected with different wolbachia strains, or reinforce an existing divergence by selecting for pre-mating isolation mechanisms
<cit> .
the presence of closely related host taxa within the cx.
pipiens complex is thus a relevant system to test such a hypothesis.
here we have approached this issue by undertaking an extensive screening for the presence, the diversity and the partitioning of wpip infections in natural populations of the four main members of the cx.
pipiens complex , spanning  <dig> natural populations and  <dig> laboratory lines.
with this aim, we characterized each wpip individual infection using two to five wolbachia markers and identified cx.
pipiens members using diagnostic nuclear markers, including microsatellites for a subsample of populations.
since wolbachia and host mitochondria are co-transmitted in egg cytoplasm and therefore are in linkage disequilibrium, we also examined the mitochondrial  diversity through the sequencing of one to three mtdna markers.
using this approach, we thus attempted to infer the contribution of wolbachia in shaping the genetic diversity within the cx.
pipiens complex.
methods
mosquito collection
we examined mosquitoes from europe, asia, oceania, africa and america belonging to the four members of the cx.
pipiens complex: cx.
p. pipiens form pipiens, cx.
p. pipiens form molestus, cx.
p. pallens as well as cx.
quinquefasciatus, hereinafter respectively referred to as pipiens, molestus, pallens and quinquefasciatus .
the collection encompasses both natural populations  and isofemale laboratory lines .
each laboratory line descended from a single female founder and was further considered as a single individual.
all specimens were stored in liquid nitrogen or in 70-95% ethanol, at room temperature or in a freezer at −20°c until examined.
molecular typing
the wpip infections were genotyped and assigned to one group  using a series of specific pcr-rflp  assays based on two ank wolbachia markers, ank <dig> and pk <dig> .
hinfi digestion of the ank <dig> pcr products allowed discrimination of five alleles : a , b , c , d  and e .
taqi digestion of the pk <dig> pcr products allowed discrimination of four specific wpip alleles : a/e , b , c  and d .
the pk <dig> a and e alleles were next distinguished using a digestion of the pk <dig> pcr products with psti: a  and e .
for a subsample of specimens, three additional wolbachia markers were sequenced: the dna mismatch repair protein gene mutl, the putative secreted protein gene gp <dig>  and the regulatory protein gene repa .
four  of these five wolbachia genes were present in one single copy in the wpip genome while the fifth  is present in three identical copies .
a total of  <dig> wolbachia genes, encompassing  <dig> distinct loci with a wide distribution along the wpip chromosome, were thus examined.
none of these genes was amplified from wolbachia-free cx.
pipiens lines, which confirmed their wolbachia origin.
dna was extracted from individual mosquitoes using a cetyltrimethylammonium bromide
<cit>  or phenol/chloroform protocols
<cit> .
all pcr amplification conditions were: 5 min at 94°c, followed by 30–40 cycles of 94°c for 30s, 50°c-58°c for 30s, and 72°c for  <dig> to  <dig>  min depending on the fragment size .
digestion of pcr products were performed following manufacturer’s instructions.
the qiaquick gel extraction kit  was used to purify the pcr products for sequencing.
sequences were obtained directly from purified products on an abi prism  <dig> sequencer using the bigdye terminator kit .
the cx.
pipiens mtdna haplotypes were determined through the sequencing of an 852 bp fragment from the cytochrome b  gene .
for a subsample of specimens, we also obtained partial sequences of two additional mtdna markers: the nadh dehydrogenase subunit  <dig>  and  <dig>  .
the four cx.
pipiens taxa were identified using nuclear dna of specimens.
we included in this study some populations available from previous studies for which cx.
pipiens taxa were determined using  <dig> to  <dig> microsatellite loci and/or sequence variation in an intron of the acetylcholinesterase- <dig>  gene .
the taxa of new cx.
pipiens populations were identified using a combination of the pcr-rflp on ace- <dig> <cit>  and a multiplex-pcr assay based on the flanking region of a microsatellite locus  .
specimens from these new populations were considered as hybrids when they showed a heterozygous genotype at ace- <dig> or cq <dig> loci.
data analyses
sequence alignments were carried out using clustalw
<cit>  and corrected using mega
<cit> .
the gblocks program
<cit>  with default parameters was used to remove poorly aligned positions and to obtain unambiguous sequence alignments.
the evolutionary model most closely fitting the sequence data was determined using akaike information criterion with the mega program
<cit> .
phylogenetic analyses were conducted using maximum likelihood  and maximum-parsimony  in mega
<cit> .
ml phylogenies were constructed based upon unambiguously aligned sites using the tajima-nei model of nucleotide substitution, assuming that nucleotide frequencies deviate substantially from  <dig>
<cit> .
mp phylogenies were constructed using the close-neighbour-interchange method
<cit> .
bootstrap probabilities were calculated by generating  <dig> bootstrap replicates.
new sequence data were deposited in genbank .
the microsatellite data was first examined for compliance with hardy–weinberg equilibrium, and then pair-wise fst values and their significance were obtained using genepop v <dig>
<cit> .
we assigned specimens to genetic clusters with a maximum likelihood algorithm implemented in the program structure  <dig> .4
<cit> .
this method combines all the individual multilocus genotypes and separates them into k distinct clusters.
we used the admixture model taking into account correlated allele frequencies between populations with  <dig>  burn-in steps and  <dig>  runs as mcmc  parameters to assign specimens to genetic clusters.
the degree of admixture, alpha, was inferred from the data .
with this parameter set, the geographic origin of each specimen is not considered but the number of clusters  is decided a priori for each run.
to assess the consistency of the analysis we performed an exhaustive comparison of  <dig> runs for each k to assess the robustness of the results.
we also performed the method described in evanno et al.
<cit>  to confirm that the true number of clusters explaining our data was detected.
the significance of non-random associations between wpip/mtdna, wpip/culex pipiens taxa or mtdna/culex pipiens taxa was estimated using an exact test procedure  implemented in genepop
<cit> .
RESULTS
polymorphism of wpip infections
we assayed for the presence and the diversity of wolbachia in  <dig> specimens from  <dig> natural populations and  <dig> isofemale laboratory lines .
specific ank <dig> and pk <dig> pcr assays indicated the occurrence of infection by wolbachia in all the examined specimens, showing that infection is fixed in all cx.
quinquefasciatus, cx.
p. pipiens form pipiens, cx.
p. pipiens form molestus and cx.
p. pallens populations examined here.
we further used the ank <dig> and pk <dig> allelic profiles to assign a wpip group to each specimen .
we found in our collection the five known ank <dig> alleles and the five known pk <dig> alleles.
tests for intergenic recombination revealed significant linkage disequilibrium  for ank <dig> and pk <dig> : alleles at these loci are not randomly associated showing that they are stably co-transmitted within the wpip chromosome.
hence, the ank <dig> and pk <dig> allelic profiles are congruent and allowed unambiguously attributing the wpip group to  <dig>  of  <dig> specimens .
for the  <dig>  remaining specimens, ank <dig> and pk <dig> allelic profiles were not congruent with known haplotypes , suggesting that these two loci had undergone recombination.
to assign these  <dig> specimens to a wpip group, three additional wolbachia markers were then sequenced: mutl, gp <dig> and repa.
three to thirteen alleles were detected for each marker and led to the identification of  <dig> new wpip haplotypes from the  <dig> unassigned specimens .
some of these alleles are null alleles: some specimens did not amplify at one of the loci  showing that their wpip infections were genetically distinct from other known haplotypes.
positive amplifications of other loci  from samples with null alleles at either mutl, gp <dig> or repa loci indicate satisfactory dna template quality in these cases.
phylogenetic tree using ank <dig>  pk <dig>  mutl, gp <dig> and repa concatenated sequences  showed that almost all new wpip haplotypes fall into one of the five wpip groups .
overall, the use of the three additional wolbachia markers allowed the assignment to a wpip group to  <dig> of the  <dig> remaining specimens, with only two haplotypes  remaining not assigned.
new wpip haplotypes are identified by #number.
allelic profiles for gp <dig>  mutl and repa loci were determined for all new wpip haplotypes and for a subsample of specimens for the known wpip haplotypes.
nd, allele not determined ; dash, null allele ; asterisk, new allele.
1: table s <dig>
taking into account pcr-rflp and phylogenetic grouping, we thus found that  <dig> specimens  were infected by the wpip-i group,  <dig>  by wpip-ii,  <dig>  by wpip-iii,  <dig>  by wpip-iv,  <dig>  by wpip-v and  <dig>  by undetermined wpip groups .
we found no evidence of specimens infected by more than one wpip group: only one allele per gene was observed for each dna sample.
the subsequent sequencing of ank <dig> and pk <dig> pcr products obtained from a subsample of specimens confirmed the observed rflp profiles, with no double peaks  in electropherograms.
geographic distribution of wpip diversity
the diversity of wpip showed an important spatial variation over the distribution area of the cx.
pipiens complex .
two wpip groups were found to dominate wide geographic regions: only wpip-i was found in sub-saharan africa, south america and southeast asia, and only wpip-iii was observed in north america.
the three other groups, wpip-ii, wpip-iv and wpip-v, were less common.
while wpip-ii and wpip-v were confined to western europe and asia, respectively, with very few exceptions, wpip-iv was more disseminated and was sporadically found in europe, north africa and asia.
generally, only one wpip group was found per geographic region indicating a regional homogeneity of wolbachia infections , but a contrasting picture emerged in europe where all five wpip groups were found .
given that europe is oversampled  relative to other parts of the world, the wpip diversity observed there could be simply a function of a higher degree of sampling effort.
however, some regions have been also extensively sampled and still revealed fewer wpip groups .
thus, oversampling does not seem to be a likely explanation for the great wpip diversity found in europe.
1: table s <dig>  bars and dots represent natural populations and laboratory strains, respectively.
the bars show the prevalence of wpip group: blue, wpip-i infection; green, wpip-ii; yellow, wpip-iii; pink, wpip-iv; orange, wpip-v; black, undetermined group.
on figure
2a, culex pipiens pipiens form pipiens and culex pipiens pipiens form molestus = light blue; cx.
p. pallens = light green and cx.
quinquefasciatus = light pink .
on figure
2b, both cx.
p. pipiens form pipiens and cx.
p. pipiens form molestus are widespread in europe.
details on sample size, prevalence and cx.
pipiens complex taxa are given in additional file
1: table s <dig>
in most cases, only one wpip group was detected per population:  <dig>  of the  <dig> natural populations harboured only one wpip group while, in the remaining  <dig> , two to three wpip groups per population were observed.
twenty-two of the  <dig> populations were located in europe and half  harboured cx.
pipiens individuals infected either by wpip-ii or wpip-iii.
aside from the wpip-ii/wpip-iii mixed populations found in western europe, at least five other geographic contact zones between wpip groups exist: in the north  and in the south of italy , in north africa , in south america  and eastern asia  .
association of wpip groups with mtdna and members of the cx.
pipiens complex
to investigate the association between wpip groups and mtdna variation, we sequenced the cytb gene  in a subsample of  <dig> specimens from  <dig> cx.
pipiens natural populations and  <dig> isofemale laboratory lines.
the cytb gene displayed low variability and eleven haplotypes with only ten variable nucleotide positions throughout the 852 bp cytb fragment  were obtained .
pairwise tests revealed significant ld for wpip groups and cytb haplotypes  with a clear pattern of cytb haplotype specificity to wpip groups .
according to this association and following previous study
<cit> , cytb haplotypes were partitioned in five mtdna groups .
when analysing the congruence between the five mtdna and wpip groups, very little incongruence were observed.
for instance, although the mtdna- <dig> group contains  <dig> wpip-i-infected specimens, seven specimens infected with wpip-v were also found in this group .
a very similar pattern was found for five other specimens: one wpip-i-infected specimen was found in the mtdna- <dig> group , one wpip-ii-infected specimen in the mtdna- <dig> group , and  <dig> wpip-iii-infected specimens in the mtdna- <dig> group .
to improve our understanding of this incongruence, two additional mtdna genes  were sequenced and phylogenetic analyses were conducted using cytb, nd <dig> and nd <dig> concatenated genes .
only the seven wpip-v-infected specimens found associated with the mtdna- <dig> group on the basis of cytb sequence were analysed since not enough dna was available from the five remaining specimens.
these analyses also included the two unassigned wpip haplotypes  described above.
the resulting phylogenetic tree does not group the seven incongruent specimens infected by wpip-v within the extended mtdna- <dig> group, and they can not be assigned to a specific mtdna group.
these results suggest that the primary wpip-mtdna incongruence observed in these specimens is likely due to low polymorphism in the mitochondrial gene studied here .
the two unassigned wpip haplotypes, # <dig> and # <dig>  were clearly associated with mtdna- <dig> and mtdna- <dig>  respectively.
the strong association between mtdna and wpip infection presented above indicates that these two haplotypes could be assigned with a low risk of error to wpip-ii and wpip-iii groups.
to investigate the association of wpip groups with taxa within the cx.
pipiens complex, we examined the partitioning of wpip groups among  <dig> cx.
pipiens specimens assigned to one of four cx.
pipiens complex taxa: quinquefasciatus , pipiens , molestus , and pallens  as well as a few hybrids  .
the observed geographic distribution of our specimens was very similar to the known distribution of the cx.
pipiens taxa
<cit>  with quinquefasciatus widely present in tropical areas, pipiens and molestus in europe, north africa, middle east and north america, and pallens confined to east asia .
the association between cx.
pipiens taxa and wpip groups was highly significant , as shown with quinquefasciatus that appeared mainly infected with the wpip-i group , pipiens with wpip-ii  and pallens with wpip-v  .
however, the pattern is less clear for molestus that showed a more balanced prevalence of wpip groups with wpip-i, wpip-ii and wpip-iii more or less equally prevalent in molestus .
overall, the association wpip/culex pipiens taxa is thus far from exclusive since no wpip group was unique to a particular cx.
pipiens member: each cx.
pipiens taxa harbours two to five different wpip groups.
for instance, pipiens harbours infections of all five wpip groups and quinquefasciatus of three wpip groups .
alternatively, the heterogeneity of wpip infections among cx.
pipiens members could be based on potential artefacts from molecular typing: for some samples, taxa identification was done through the characterization of two nuclear markers, ace- <dig> and cq <dig>  an approach that could fail to detect hybrid individuals.
to address this potential bias we refined our analysis by restricting our analyses to a subsample of  <dig> specimens from  <dig> populations  that were typed with microsatellite loci.
although these mosquito populations were originally typed using 7– <dig> microsatellite loci, the loci used were however different depending on what cx.
pipiens taxa and studies they were for.
further analyses were thus conducted using four microsatellite loci  for which genotypes were available for all specimens.
none of these loci had significant heterozygote deficits/excess in the examined populations, fitting with hardy–weinberg assumptions.
a pair-wise fst comparison revealed significant differentiation between mosquito populations belonging to different taxa in all cases , while within taxa the fst values ranged from only  <dig>  to  <dig>  and were in some cases not different from zero , indicating that gene flow is more important within than between cx.
pipiens taxa.
the result of the mosquito genetic structure analysis further separates the individuals into five distinct clusters analogous to the culex taxa assignation .
indeed, the clustering separate pallens, pipiens, molestus, quinquefasciatus from america and quinquefasciatus from africa and asia.
this clustering indicates a low hybrid rate  indicating that most individuals are not admixed and come from a cluster .
the assignment of individuals using microsatellite data was in agreement with our primary assignment, although it separates quinquefasciatus populations in two distinct clusters depending on their geographic origins.
same as above, the association between wpip groups and cx.
pipiens members was still significant  and also confirmed that this association is not exclusive since at least four wpip groups are shared by different cx.
pipiens members .
the presence of shared wpip groups between cx.
pipiens members thus suggests that wpip infections undergo repeated transfers between quinquefasciatus, pipiens, molestus and pallens.
1: table s <dig>
finally, the association between mtdna and cx.
pipiens members was assayed in  <dig> individuals .
as observed with wpip groups, there was a significant association between mtdna and cx.
pipiens members , but not exclusive: although mtdna- <dig> group was common in quinquefasciatus  and mtdna- <dig> group was common in pipiens , no mtdna group was specific to a particular cx.
pipiens member .
as could be expected due to ld between wpip and mtdna, the mtdna distribution thus mirrored the wpip distribution across the cx.
pipiens complex, showing that they form together a single cytoplasmic unit.
overall, these results show that wpip and mtdna share a joint evolutionary history, subtly different to the one of nuclear dna and thus to the evolutionary histories of cx.
pipiens members.
discussion
we sampled four evolutionarily closely related mosquito taxa within the cx.
pipiens complex for wolbachia infection study.
we further specifically tested for host-specific associations by characterizing wolbachia and mtdna haplotypes using multilocus typing schemes.
the observed prevalence of wpip infection was 100% in all cx.
quinquefasciatus, cx.
p. pipiens form pipiens, cx.
p. pipiens form molestus and cx.
p. pallens populations, as usually recorded for these cx.
pipiens taxa
<cit> .
the genotyping of wpip strains using the two ank genes ank <dig> and pk1confirmed the presence of five distinct wpip groups as observed in a previous study
<cit>  showing that ank <dig> and pk <dig> genes are suitable to assign wpip groups.
however, by using additional wpip markers we detected greater diversity including new and recombinant haplotypes indicating higher wpip diversity than previously thought.
overall, our survey of infection diversity is therefore likely to seriously underestimate the true figure of wpip diversity that shows a rapid diversification in their natural host cx.
pipiens.
the distribution of wpip groups appeared spatially structured, well exemplified by european cx.
pipiens populations that harbour the highest wpip diversity across all examined geographic regions.
this suggests that the wpip ancestor may have initially spread in european populations where it evolved in five divergent groups, and that newly emerged wpip groups have only secondarily expanded outside europe.
human migrations have also probably enhanced this process by expanding the geographic range of diverse cx.
pipiens members.
for instance, both cx.
pipiens and cx.
quinquefasciatus were recently introduced into the americas and australia
<cit> .
in the americas, the presence of two wpip groups indicates that at least two separate introductions have occurred: one probably from europe introduced wpip-iii-infected cx.
pipiens to north america, the other from tropical africa or south asia introduced wpip-i-infected cx.
quinquefasciatus to americas.
in australia, independent introduction events may also explain the presence of wpip-ii and wpip-v, otherwise mainly found in western europe and asia.
that american and australian populations came from multiple and independent colonization events from europe, africa and asia is also well supported by the examination of cx.
pipiens microsatellites
<cit> .
overall, the wpip distribution is likely to result from ancient and recent imprints, underlining the importance of historical contingencies in the population structure of infections.
remarkably, the mtdna variation in the cx.
pipiens complex mirrored precisely the wpip variation, showing a great evidence for indirect selection arising from linkage disequilibrium with infections.
mtdna diversity was extremely reduced over the distribution area of cx.
pipiens members, which is likely to be a consequence of cytoplasmic hitchhiking driven by the recent invasion of the wpip ancestor, as pointed by previous studies
<cit> .
the mtdna of cx.
pipiens individuals infected by different wpip groups have further evolved to be distinct, showing that these two cytoplasmic elements have experienced a recent joint evolutionary history.
as a result, wpip confounds the inference of cx.
pipiens evolutionary history from mtdna data, as often observed in other wolbachia-infected species
<cit> .
because of vertical transmission, one should expect that persistence of wpip infection in the cx.
pipiens complex over long periods of time should result in diversification of wolbachia alongside the host .
however, we did not observe this pattern: similar wpip groups and mtdna haplotypes are found in different taxa and no cytoplasm type is specific to a given cx.
pipiens taxa.
we rather showed that the cytoplasmic diversity tends to be homogenized across the four members of the cx.
pipiens complex we examined.
collectively, the data suggest a recent, and possibly still ongoing, cytoplasmic exchange between cx.
pipiens taxa.
two non-exclusive processes can explain why different cx.
pipiens taxa share similar cytoplasms.
the first process is the relatively recent emergence of some cx.
pipiens taxa.
the pipiens and molestus forms are very closely related and several scenarios place their divergence at  <dig>  years ago
<cit> , which is slightly more recent than the supposed emergence of wpip groups .
assuming that wpip infections were initially present in the pipiens-molestus ancestor could explain why many shared wpip groups were found in both cx.
pipiens taxa.
the second process that could lead the homogenization of cytoplasmic diversity is linked to hybridization events within the complex.
although each member of the cx.
pipiens complex has an unique genetic signature, their genetic independence is not absolute since occasional inter-taxa hybridization may occur .
hybridization may transfer wpip infections and associated mtdna from taxa to taxa
<cit> .
this pattern fits well with the case of cx.
quinquefasciatus.
this taxon has emerged long before the pipiens-molestus divergence
<cit> , and before the initial wpip infection in european populations.
it is thus likely that wpip has secondarily spread from cx.
pipiens to cx.
quinquefasciatus following cytoplasmic introgression since recurring hybridization occurs where their distribution overlaps
<cit> .
a similar process could also explain the presence of two different wpip groups in cx.
p. pallens: this latter taxa is thought to be the result of unidirectional hybridization between cx.
p. pipiens females and cx.
quinquefasciatus males
<cit> , a pattern that explains why cx.
p. pipiens and cx.
p. pallens share two wpip groups.
cytoplasmic introgression is likely still underway in the cx.
pipiens complex: hybrids between pipiens and molestus forms have been documented in north america and in southern europe
<cit>  as well as between cx.
quinquefasciatus and cx.
p. pallens in asia
<cit> , and wpip infections may commonly flow from either side into the hybrid zone.
CONCLUSIONS
it is now clear that wolbachia impacts the cx.
pipiens diversity in different ways.
the wpip distribution presented here showed a well-structured picture, and underlines a critical example of cytoplasmic introgression through sibling taxa.
this situation is actually similar to adaptive introgression of beneficial alleles.
previous studies examining loci involved in insecticide resistance concluded that resistant alleles are undergoing frequent expansion across the complex through hybridization between cx.
pipiens members
<cit> .
survey of cx.
pipiens neutral loci, however, show that recombination may break the connection between selected and neutral loci very quickly, maintaining the genetic differentiation between taxa and allowing the inter-taxa expansion of selected genes
<cit> .
hybridization can weakly impact the global flow of nuclear genes but serves as a powerful mechanism of rapid adaptation for insect populations through the penetration of useful adaptive alleles.
the wpip infections seem to operate in a similar manner as insecticide resistance genes in the cx.
pipiens members, suggesting that introgression has a crucial role in the dispersal of wpip infections.
of note, however, in the case of wolbachia, infection spread may have a negative impact on population dynamics of mosquito hosts since it could drive deleterious mitotypes to fixation through cytoplasmic hitchhiking.
it remains to be seen what role, if any, ci between incompatible wpip strains play in divergence between different geographic populations within the cx.
pipiens complex.
competing interests
the authors declare that they have no competing interests.
authors’ contributions
conceived and designed the experiments: ed, cma, mw and od.
performed the experiments: ed, cma, od, p makoundou, su, dmf, evs.
analyzed the data: ed, p milesi and od.
wrote the paper: ed, mw and od.
all authors read and approved the final manuscript.
supplementary material
additional file 1: table s1
detailed results of the screen of culex pipiens populations.
ll, laboratory lines; nd, not determined.
table s <dig>  list of primers and gene features.
table s <dig>  nucleotide polymorphism in the cytb, nd <dig> and nd <dig> mitochondrial genes.
only polymorphic sites are represented and a dash indicates similarity with the top sequence.
position expressed in nucleotides bases on the complete mitochondrial sequence of pel culex pipiens line .
table s <dig>  pair-wise fst values among mosquito populations .
*, significant fst values after bonferroni correction.
figure s <dig>  identification of ank <dig> and pk <dig> allelic profiles.
hinfi digestion of the ank <dig> pcr products allowed discrimination of five alleles : a , b , c , d  and e .
taqi digestion of the pk <dig> pcr products allowed discrimination of four specific wpip alleles : a/e , b , c  and d .
psti digestion of the pk <dig> pcr products allowed discrimination alleles a  and e .
figure s <dig> mtdna phylogeny constructed using maximum likelihood method based on concatenated sequences of cytb, nd <dig> and nd <dig> genes.
mtdna haplotypes originally described by atyame et al.
are marked by full circles.
triangles show the seven specimens presenting incongruences between wpip infection and mtdna haplotypes.
numbers on branches indicate percentage bootstrap support for major branches .
the scale bar indicates the number of substitutions.
click here for file
acknowledgments
we are very grateful to n. pasteur, f. zélé, f. tredez, j. tonnabelle, s. boukraa, c. malcolm, m. raymond, f. rousset, n. perrin, r. dabire, g. duvallet, p. tortosa, p. carnevale, p. guillet, c. ngoagouni, i. dufour, j. vontas, a. tsagkarakou, d. ayala, m calviti, m. toure, f. cui, s. marcombe and g. müller for collection help.
we are also grateful to n. pasteur and p. labbé, for their helpful comments and suggestions, and to a. berthomieu for technical help.
all sequence data were obtained on the environmental genomic platform of the ifr montpellier-environnement-biodiversité.
contribution 2013-x of the institut des sciences de l'evolution de montpellier .

 symbiotic associations with the intracellular bacterium wolbachia are extremely widespread in insects. wolbachia is typically maternally inherited through the egg cytoplasm and has evolved a variety of interactions with its hosts, exerting subtle effects such as manipulation of host reproduction or protection against natural enemies. each of these effects is advantageous to infected females and thus enables wolbachia to spread rapidly through insect populations. these effects are also of ecological and evolutionary importance to the particular host species that is infected, potentially inducing reproductive isolation or driving changes in sexuality. mosquitoes of the culex pipiens complex have long been recognized to exhibit a great variability of effects associated with wolbachia. in this host, wolbachia, known as wpip, is associated with cytoplasmic incompatibility, a sperm-egg incompatibility between infected males and uninfected females, so that infected females have a reproductive advantage. apart from this simple case, ci has been also observed in a number of cases between males and females carrying incompatible wpip strains. five housekeeping genes developed for wolbachia multilocus strain typing and the wolbachia surface protein gene wsp present no variation among these wpip strains, showing that they delineate a recent and monophyletic clade into the b wolbachia supergroup. the recent examination of fast evolving markers, such as the ank and pk genes encoding proteins with ankyrin motifs, revealed the existence of more than genetically distinct wpip strains, belonging to five distinct subclades and further referred as wpip-i to wpip-v groups. since their molecular characterization, several recent studies have further showed that some wpip strains are mutually incompatible but also that some others, although genetically distinct, are fully compatible. meanwhile, wpip has also emerged as a conditional mutualist that protects cx. pipiens against mortality induced by the avian malaria parasite plasmodium relictum. overall, these studies revealed that wpip is an important associate of most mosquitoes in the cx. pipiens complex potentially driving its evolution. how wpip infections interact with the genetic structure within the cx. pipiens complex remains still unclear. mosquitoes in this complex have a global distribution in all temperate and tropical regions, with a recent history of association with human migration. the complex encompasses a group of genetically closely related taxa with distinct behavioural and physiological traits that greatly influence their distribution. the most obvious variable traits include larval habitat preference, vertebrate feeding pattern, mating behaviour, gonotrophic development and ability to enter into diapause during the winter. despite these differences, members of the cx. pipiens complex have controversial taxonomic statuses and, even though they are genetically distinct, they remain difficult to separate morphologically. four species, or subspecies according to the authors, are frequently recognized as members of the complex: cx. pipiens, cx. quinquefasciatus, cx. australicus, and cx. globocoxitus, as well as two subspecies: cx. pipiens pipiens in europe and north and south africa, and cx. p. pallens, in asia. in addition, two sympatric forms, pipiens and molestus, are also encountered in cx. p. pipiens in the northern hemisphere. cx. quinquefasciatus, commonly known as the southern house mosquito, exists all across the tropics and the lower latitudes of temperate regions. cx. australicus and cx. globocoxitus are restricted to australia and are poorly known. although the exact taxonomic status of the cx. pipiens members remains controversial, their close evolutionary association has been repeatedly supported by genetic analyses and the relative abundance of hybrids in areas where distributions overlap. remarkably, whilst wpip was never detected in cx. australicus and cx. globocoxitus, infection frequency was near or at fixation in almost all populations of both cx. pipiens and cx. quinquefasciatus. when first described, any of the five wpip groups were found associated within given infected members of the cx. pipiens complex. however, only a limited number of cx. pipiens and cx. quinquefasciatus laboratory lines have been examined, making this result difficult to interpret. this issue is of special importance since wolbachia may either produce reproductive isolation between host populations infected with different wolbachia strains, or reinforce an existing divergence by selecting for pre-mating isolation mechanisms. the presence of closely related host taxa within the cx. pipiens complex is thus a relevant system to test such a hypothesis. here we have approached this issue by undertaking an extensive screening for the presence, the diversity and the partitioning of wpip infections in natural populations of the four main members of the cx. pipiens complex, spanning natural populations and laboratory lines. with this aim, we characterized each wpip individual infection using two to five wolbachia markers and identified cx. pipiens members using diagnostic nuclear markers, including microsatellites for a subsample of populations. since wolbachia and host mitochondria are co-transmitted in egg cytoplasm and therefore are in linkage disequilibrium, we also examined the mitochondrial diversity through the sequencing of one to three mtdna markers. using this approach, we thus attempted to infer the contribution of wolbachia in shaping the genetic diversity within the cx. pipiens complex. methods mosquito collection we examined mosquitoes from europe, asia, oceania, africa and america belonging to the four members of the cx. pipiens complex: cx. p. pipiens form pipiens, cx. p. pipiens form molestus, cx. p. pallens as well as cx. quinquefasciatus, hereinafter respectively referred to as pipiens, molestus, pallens and quinquefasciatus. the collection encompasses both natural populations and isofemale laboratory lines. each laboratory line descended from a single female founder and was further considered as a single individual. all specimens were stored in liquid nitrogen or in 70-95% ethanol, at room temperature or in a freezer at −20°c until examined. molecular typing the wpip infections were genotyped and assigned to one group using a series of specific pcr-rflp assays based on two ank wolbachia markers, ank and pk. hinfi digestion of the ank pcr products allowed discrimination of five alleles: a, b, c, d and e. taqi digestion of the pk pcr products allowed discrimination of four specific wpip alleles: a/e, b, c and d. the pk a and e alleles were next distinguished using a digestion of the pk pcr products with psti: a and e. for a subsample of specimens, three additional wolbachia markers were sequenced: the dna mismatch repair protein gene mutl, the putative secreted protein gene gp and the regulatory protein gene repa. four of these five wolbachia genes were present in one single copy in the wpip genome while the fifth is present in three identical copies. a total of wolbachia genes, encompassing distinct loci with a wide distribution along the wpip chromosome, were thus examined. none of these genes was amplified from wolbachia-free cx. pipiens lines, which confirmed their wolbachia origin. dna was extracted from individual mosquitoes using a cetyltrimethylammonium bromide or phenol/chloroform protocols. all pcr amplification conditions were: 5 min at 94°c, followed by 30–40 cycles of 94°c for 30s, 50°c-58°c for 30s, and 72°c for to  min depending on the fragment size. digestion of pcr products were performed following manufacturer’s instructions. the qiaquick gel extraction kit was used to purify the pcr products for sequencing. sequences were obtained directly from purified products on an abi prism sequencer using the bigdye terminator kit. the cx. pipiens mtdna haplotypes were determined through the sequencing of an 852 bp fragment from the cytochrome b gene. for a subsample of specimens, we also obtained partial sequences of two additional mtdna markers: the nadh dehydrogenase subunit and. the four cx. pipiens taxa were identified using nuclear dna of specimens. we included in this study some populations available from previous studies for which cx. pipiens taxa were determined using to microsatellite loci and/or sequence variation in an intron of the acetylcholinesterase- gene. the taxa of new cx. pipiens populations were identified using a combination of the pcr-rflp on ace- and a multiplex-pcr assay based on the flanking region of a microsatellite locus. specimens from these new populations were considered as hybrids when they showed a heterozygous genotype at ace- or cq loci. data analyses sequence alignments were carried out using clustalw and corrected using mega. the gblocks program with default parameters was used to remove poorly aligned positions and to obtain unambiguous sequence alignments. the evolutionary model most closely fitting the sequence data was determined using akaike information criterion with the mega program. phylogenetic analyses were conducted using maximum likelihood and maximum-parsimony in mega. ml phylogenies were constructed based upon unambiguously aligned sites using the tajima-nei model of nucleotide substitution, assuming that nucleotide frequencies deviate substantially from. mp phylogenies were constructed using the close-neighbour-interchange method. bootstrap probabilities were calculated by generating bootstrap replicates. new sequence data were deposited in genbank. the microsatellite data was first examined for compliance with hardy–weinberg equilibrium, and then pair-wise fst values and their significance were obtained using genepop v. we assigned specimens to genetic clusters with a maximum likelihood algorithm implemented in the program structure.4. this method combines all the individual multilocus genotypes and separates them into k distinct clusters. we used the admixture model taking into account correlated allele frequencies between populations with burn-in steps and runs as mcmc parameters to assign specimens to genetic clusters. the degree of admixture, alpha, was inferred from the data. with this parameter set, the geographic origin of each specimen is not considered but the number of clusters is decided a priori for each run. to assess the consistency of the analysis we performed an exhaustive comparison of runs for each k to assess the robustness of the results. we also performed the method described in evanno et al. to confirm that the true number of clusters explaining our data was detected. the significance of non-random associations between wpip/mtdna, wpip/culex pipiens taxa or mtdna/culex pipiens taxa was estimated using an exact test procedure implemented in genepop.  polymorphism of wpip infections we assayed for the presence and the diversity of wolbachia in specimens from natural populations and isofemale laboratory lines. specific ank and pk pcr assays indicated the occurrence of infection by wolbachia in all the examined specimens, showing that infection is fixed in all cx. quinquefasciatus, cx. p. pipiens form pipiens, cx. p. pipiens form molestus and cx. p. pallens populations examined here. we further used the ank and pk allelic profiles to assign a wpip group to each specimen. we found in our collection the five known ank alleles and the five known pk alleles. tests for intergenic recombination revealed significant linkage disequilibrium for ank and pk: alleles at these loci are not randomly associated showing that they are stably co-transmitted within the wpip chromosome. hence, the ank and pk allelic profiles are congruent and allowed unambiguously attributing the wpip group to of specimens. for the remaining specimens, ank and pk allelic profiles were not congruent with known haplotypes, suggesting that these two loci had undergone recombination. to assign these specimens to a wpip group, three additional wolbachia markers were then sequenced: mutl, gp and repa. three to thirteen alleles were detected for each marker and led to the identification of new wpip haplotypes from the unassigned specimens. some of these alleles are null alleles: some specimens did not amplify at one of the loci showing that their wpip infections were genetically distinct from other known haplotypes. positive amplifications of other loci from samples with null alleles at either mutl, gp or repa loci indicate satisfactory dna template quality in these cases. phylogenetic tree using ank pk mutl, gp and repa concatenated sequences showed that almost all new wpip haplotypes fall into one of the five wpip groups. overall, the use of the three additional wolbachia markers allowed the assignment to a wpip group to of the remaining specimens, with only two haplotypes remaining not assigned. new wpip haplotypes are identified by #number. allelic profiles for gp mutl and repa loci were determined for all new wpip haplotypes and for a subsample of specimens for the known wpip haplotypes. nd, allele not determined; dash, null allele; asterisk, new allele. 1: table s taking into account pcr-rflp and phylogenetic grouping, we thus found that specimens were infected by the wpip-i group, by wpip-ii, by wpip-iii, by wpip-iv, by wpip-v and by undetermined wpip groups. we found no evidence of specimens infected by more than one wpip group: only one allele per gene was observed for each dna sample. the subsequent sequencing of ank and pk pcr products obtained from a subsample of specimens confirmed the observed rflp profiles, with no double peaks in electropherograms. geographic distribution of wpip diversity the diversity of wpip showed an important spatial variation over the distribution area of the cx. pipiens complex. two wpip groups were found to dominate wide geographic regions: only wpip-i was found in sub-saharan africa, south america and southeast asia, and only wpip-iii was observed in north america. the three other groups, wpip-ii, wpip-iv and wpip-v, were less common. while wpip-ii and wpip-v were confined to western europe and asia, respectively, with very few exceptions, wpip-iv was more disseminated and was sporadically found in europe, north africa and asia. generally, only one wpip group was found per geographic region indicating a regional homogeneity of wolbachia infections, but a contrasting picture emerged in europe where all five wpip groups were found. given that europe is oversampled relative to other parts of the world, the wpip diversity observed there could be simply a function of a higher degree of sampling effort. however, some regions have been also extensively sampled and still revealed fewer wpip groups. thus, oversampling does not seem to be a likely explanation for the great wpip diversity found in europe. 1: table s bars and dots represent natural populations and laboratory strains, respectively. the bars show the prevalence of wpip group: blue, wpip-i infection; green, wpip-ii; yellow, wpip-iii; pink, wpip-iv; orange, wpip-v; black, undetermined group. on figure  2a, culex pipiens pipiens form pipiens and culex pipiens pipiens form molestus = light blue; cx. p. pallens = light green and cx. quinquefasciatus = light pink. on figure  2b, both cx. p. pipiens form pipiens and cx. p. pipiens form molestus are widespread in europe. details on sample size, prevalence and cx. pipiens complex taxa are given in additional file 1: table s in most cases, only one wpip group was detected per population: of the natural populations harboured only one wpip group while, in the remaining, two to three wpip groups per population were observed. twenty-two of the populations were located in europe and half harboured cx. pipiens individuals infected either by wpip-ii or wpip-iii. aside from the wpip-ii/wpip-iii mixed populations found in western europe, at least five other geographic contact zones between wpip groups exist: in the north and in the south of italy, in north africa, in south america and eastern asia. association of wpip groups with mtdna and members of the cx. pipiens complex to investigate the association between wpip groups and mtdna variation, we sequenced the cytb gene in a subsample of specimens from cx. pipiens natural populations and isofemale laboratory lines. the cytb gene displayed low variability and eleven haplotypes with only ten variable nucleotide positions throughout the 852 bp cytb fragment were obtained. pairwise tests revealed significant ld for wpip groups and cytb haplotypes with a clear pattern of cytb haplotype specificity to wpip groups. according to this association and following previous study, cytb haplotypes were partitioned in five mtdna groups. when analysing the congruence between the five mtdna and wpip groups, very little incongruence were observed. for instance, although the mtdna- group contains wpip-i-infected specimens, seven specimens infected with wpip-v were also found in this group. a very similar pattern was found for five other specimens: one wpip-i-infected specimen was found in the mtdna- group, one wpip-ii-infected specimen in the mtdna- group, and wpip-iii-infected specimens in the mtdna- group. to improve our understanding of this incongruence, two additional mtdna genes were sequenced and phylogenetic analyses were conducted using cytb, nd and nd concatenated genes. only the seven wpip-v-infected specimens found associated with the mtdna- group on the basis of cytb sequence were analysed since not enough dna was available from the five remaining specimens. these analyses also included the two unassigned wpip haplotypes described above. the resulting phylogenetic tree does not group the seven incongruent specimens infected by wpip-v within the extended mtdna- group, and they can not be assigned to a specific mtdna group. these results suggest that the primary wpip-mtdna incongruence observed in these specimens is likely due to low polymorphism in the mitochondrial gene studied here. the two unassigned wpip haplotypes, # and # were clearly associated with mtdna- and mtdna- respectively. the strong association between mtdna and wpip infection presented above indicates that these two haplotypes could be assigned with a low risk of error to wpip-ii and wpip-iii groups. to investigate the association of wpip groups with taxa within the cx. pipiens complex, we examined the partitioning of wpip groups among cx. pipiens specimens assigned to one of four cx. pipiens complex taxa: quinquefasciatus, pipiens, molestus, and pallens as well as a few hybrids. the observed geographic distribution of our specimens was very similar to the known distribution of the cx. pipiens taxa with quinquefasciatus widely present in tropical areas, pipiens and molestus in europe, north africa, middle east and north america, and pallens confined to east asia. the association between cx. pipiens taxa and wpip groups was highly significant, as shown with quinquefasciatus that appeared mainly infected with the wpip-i group, pipiens with wpip-ii and pallens with wpip-v. however, the pattern is less clear for molestus that showed a more balanced prevalence of wpip groups with wpip-i, wpip-ii and wpip-iii more or less equally prevalent in molestus. overall, the association wpip/culex pipiens taxa is thus far from exclusive since no wpip group was unique to a particular cx. pipiens member: each cx. pipiens taxa harbours two to five different wpip groups. for instance, pipiens harbours infections of all five wpip groups and quinquefasciatus of three wpip groups. alternatively, the heterogeneity of wpip infections among cx. pipiens members could be based on potential artefacts from molecular typing: for some samples, taxa identification was done through the characterization of two nuclear markers, ace- and cq an approach that could fail to detect hybrid individuals. to address this potential bias we refined our analysis by restricting our analyses to a subsample of specimens from populations that were typed with microsatellite loci. although these mosquito populations were originally typed using 7– microsatellite loci, the loci used were however different depending on what cx. pipiens taxa and studies they were for. further analyses were thus conducted using four microsatellite loci for which genotypes were available for all specimens. none of these loci had significant heterozygote deficits/excess in the examined populations, fitting with hardy–weinberg assumptions. a pair-wise fst comparison revealed significant differentiation between mosquito populations belonging to different taxa in all cases, while within taxa the fst values ranged from only to and were in some cases not different from zero, indicating that gene flow is more important within than between cx. pipiens taxa. the result of the mosquito genetic structure analysis further separates the individuals into five distinct clusters analogous to the culex taxa assignation. indeed, the clustering separate pallens, pipiens, molestus, quinquefasciatus from america and quinquefasciatus from africa and asia. this clustering indicates a low hybrid rate indicating that most individuals are not admixed and come from a cluster. the assignment of individuals using microsatellite data was in agreement with our primary assignment, although it separates quinquefasciatus populations in two distinct clusters depending on their geographic origins. same as above, the association between wpip groups and cx. pipiens members was still significant and also confirmed that this association is not exclusive since at least four wpip groups are shared by different cx. pipiens members. the presence of shared wpip groups between cx. pipiens members thus suggests that wpip infections undergo repeated transfers between quinquefasciatus, pipiens, molestus and pallens. 1: table s finally, the association between mtdna and cx. pipiens members was assayed in individuals. as observed with wpip groups, there was a significant association between mtdna and cx. pipiens members, but not exclusive: although mtdna- group was common in quinquefasciatus and mtdna- group was common in pipiens, no mtdna group was specific to a particular cx. pipiens member. as could be expected due to ld between wpip and mtdna, the mtdna distribution thus mirrored the wpip distribution across the cx. pipiens complex, showing that they form together a single cytoplasmic unit. overall, these results show that wpip and mtdna share a joint evolutionary history, subtly different to the one of nuclear dna and thus to the evolutionary histories of cx. pipiens members. discussion we sampled four evolutionarily closely related mosquito taxa within the cx. pipiens complex for wolbachia infection study. we further specifically tested for host-specific associations by characterizing wolbachia and mtdna haplotypes using multilocus typing schemes. the observed prevalence of wpip infection was 100% in all cx. quinquefasciatus, cx. p. pipiens form pipiens, cx. p. pipiens form molestus and cx. p. pallens populations, as usually recorded for these cx. pipiens taxa. the genotyping of wpip strains using the two ank genes ank and pk1confirmed the presence of five distinct wpip groups as observed in a previous study showing that ank and pk genes are suitable to assign wpip groups. however, by using additional wpip markers we detected greater diversity including new and recombinant haplotypes indicating higher wpip diversity than previously thought. overall, our survey of infection diversity is therefore likely to seriously underestimate the true figure of wpip diversity that shows a rapid diversification in their natural host cx. pipiens. the distribution of wpip groups appeared spatially structured, well exemplified by european cx. pipiens populations that harbour the highest wpip diversity across all examined geographic regions. this suggests that the wpip ancestor may have initially spread in european populations where it evolved in five divergent groups, and that newly emerged wpip groups have only secondarily expanded outside europe. human migrations have also probably enhanced this process by expanding the geographic range of diverse cx. pipiens members. for instance, both cx. pipiens and cx. quinquefasciatus were recently introduced into the americas and australia. in the americas, the presence of two wpip groups indicates that at least two separate introductions have occurred: one probably from europe introduced wpip-iii-infected cx. pipiens to north america, the other from tropical africa or south asia introduced wpip-i-infected cx. quinquefasciatus to americas. in australia, independent introduction events may also explain the presence of wpip-ii and wpip-v, otherwise mainly found in western europe and asia. that american and australian populations came from multiple and independent colonization events from europe, africa and asia is also well supported by the examination of cx. pipiens microsatellites. overall, the wpip distribution is likely to result from ancient and recent imprints, underlining the importance of historical contingencies in the population structure of infections. remarkably, the mtdna variation in the cx. pipiens complex mirrored precisely the wpip variation, showing a great evidence for indirect selection arising from linkage disequilibrium with infections. mtdna diversity was extremely reduced over the distribution area of cx. pipiens members, which is likely to be a consequence of cytoplasmic hitchhiking driven by the recent invasion of the wpip ancestor, as pointed by previous studies. the mtdna of cx. pipiens individuals infected by different wpip groups have further evolved to be distinct, showing that these two cytoplasmic elements have experienced a recent joint evolutionary history. as a result, wpip confounds the inference of cx. pipiens evolutionary history from mtdna data, as often observed in other wolbachia-infected species. because of vertical transmission, one should expect that persistence of wpip infection in the cx. pipiens complex over long periods of time should result in diversification of wolbachia alongside the host. however, we did not observe this pattern: similar wpip groups and mtdna haplotypes are found in different taxa and no cytoplasm type is specific to a given cx. pipiens taxa. we rather showed that the cytoplasmic diversity tends to be homogenized across the four members of the cx. pipiens complex we examined. collectively, the data suggest a recent, and possibly still ongoing, cytoplasmic exchange between cx. pipiens taxa. two non-exclusive processes can explain why different cx. pipiens taxa share similar cytoplasms. the first process is the relatively recent emergence of some cx. pipiens taxa. the pipiens and molestus forms are very closely related and several scenarios place their divergence at  years ago, which is slightly more recent than the supposed emergence of wpip groups. assuming that wpip infections were initially present in the pipiens-molestus ancestor could explain why many shared wpip groups were found in both cx. pipiens taxa. the second process that could lead the homogenization of cytoplasmic diversity is linked to hybridization events within the complex. although each member of the cx. pipiens complex has an unique genetic signature, their genetic independence is not absolute since occasional inter-taxa hybridization may occur. hybridization may transfer wpip infections and associated mtdna from taxa to taxa. this pattern fits well with the case of cx. quinquefasciatus. this taxon has emerged long before the pipiens-molestus divergence, and before the initial wpip infection in european populations. it is thus likely that wpip has secondarily spread from cx. pipiens to cx. quinquefasciatus following cytoplasmic introgression since recurring hybridization occurs where their distribution overlaps. a similar process could also explain the presence of two different wpip groups in cx. p. pallens: this latter taxa is thought to be the result of unidirectional hybridization between cx. p. pipiens females and cx. quinquefasciatus males, a pattern that explains why cx. p. pipiens and cx. p. pallens share two wpip groups. cytoplasmic introgression is likely still underway in the cx. pipiens complex: hybrids between pipiens and molestus forms have been documented in north america and in southern europe as well as between cx. quinquefasciatus and cx. p. pallens in asia, and wpip infections may commonly flow from either side into the hybrid zone.  it is now clear that wolbachia impacts the cx. pipiens diversity in different ways. the wpip distribution presented here showed a well-structured picture, and underlines a critical example of cytoplasmic introgression through sibling taxa. this situation is actually similar to adaptive introgression of beneficial alleles. previous studies examining loci involved in insecticide resistance concluded that resistant alleles are undergoing frequent expansion across the complex through hybridization between cx. pipiens members. survey of cx. pipiens neutral loci, however, show that recombination may break the connection between selected and neutral loci very quickly, maintaining the genetic differentiation between taxa and allowing the inter-taxa expansion of selected genes. hybridization can weakly impact the global flow of nuclear genes but serves as a powerful mechanism of rapid adaptation for insect populations through the penetration of useful adaptive alleles. the wpip infections seem to operate in a similar manner as insecticide resistance genes in the cx. pipiens members, suggesting that introgression has a crucial role in the dispersal of wpip infections. of note, however, in the case of wolbachia, infection spread may have a negative impact on population dynamics of mosquito hosts since it could drive deleterious mitotypes to fixation through cytoplasmic hitchhiking. it remains to be seen what role, if any, ci between incompatible wpip strains play in divergence between different geographic populations within the cx. pipiens complex. competing interests the authors declare that they have no competing interests. authors’ contributions conceived and designed the experiments: ed, cma, mw and od. performed the experiments: ed, cma, od, p makoundou, su, dmf, evs. analyzed the data: ed, p milesi and od. wrote the paper: ed, mw and od. all authors read and approved the final manuscript. supplementary material additional file 1: table s1 detailed results of the screen of culex pipiens populations. ll, laboratory lines; nd, not determined. table s list of primers and gene features. table s nucleotide polymorphism in the cytb, nd and nd mitochondrial genes. only polymorphic sites are represented and a dash indicates similarity with the top sequence. position expressed in nucleotides bases on the complete mitochondrial sequence of pel culex pipiens line. table s pair-wise fst values among mosquito populations. *, significant fst values after bonferroni correction. figure s identification of ank and pk allelic profiles. hinfi digestion of the ank pcr products allowed discrimination of five alleles: a, b, c, d and e. taqi digestion of the pk pcr products allowed discrimination of four specific wpip alleles: a/e, b, c and d. psti digestion of the pk pcr products allowed discrimination alleles a and e. figure s mtdna phylogeny constructed using maximum likelihood method based on concatenated sequences of cytb, nd and nd genes. mtdna haplotypes originally described by atyame et al. are marked by full circles. triangles show the seven specimens presenting incongruences between wpip infection and mtdna haplotypes. numbers on branches indicate percentage bootstrap support for major branches. the scale bar indicates the number of substitutions. click here for file acknowledgments we are very grateful to n. pasteur, f. zélé, f. tredez, j. tonnabelle, s. boukraa, c. malcolm, m. raymond, f. rousset, n. perrin, r. dabire, g. duvallet, p. tortosa, p. carnevale, p. guillet, c. ngoagouni, i. dufour, j. vontas, a. tsagkarakou, d. ayala, m calviti, m. toure, f. cui, s. marcombe and g. müller for collection help. we are also grateful to n. pasteur and p. labbé, for their helpful comments and suggestions, and to a. berthomieu for technical help. all sequence data were obtained on the environmental genomic platform of the ifr montpellier-environnement-biodiversité. contribution 2013-x of the institut des sciences de l'evolution de montpellier.
the maternally inherited bacterium wolbachia often acts as a subtle parasite that manipulates insect reproduction, resulting potentially in reproductive isolation between host populations. this data overall supports the hypothesis that wpip infections, and their allied mitochondria, are associated with regular transfers between cx. there was a strong association between wolbachia and mitochondrial haplotypes indicating a stable co-transmission in mosquito populations. pipiens complex are genetically distinct on the basis of nuclear dna, the association of wolbachia and mtdna with members of the cx. overall, this is suggestive of a recent and likely ongoing cytoplasmic introgression through hybridization events across the cx. whilst distinct wolbachia strains are documented in a group of evolutionarily closely related mosquitoes known as the culex pipiens complex, their impact on mosquito population genetics remains unclear. four other wpip groups were also found in mosquito populations and all were shared between diverse cx. pipiens members rather than specific host associations. we further examined the wolbachia genetic diversity, the variability in the coinherited host mitochondria and their partitioning among members of the cx.
 the maternally inherited bacterium wolbachia often acts as a subtle parasite that manipulates insect reproduction, resulting potentially in reproductive isolation between host populations. whilst distinct wolbachia strains are documented in a group of evolutionarily closely related mosquitoes known as the culex pipiens complex, their impact on mosquito population genetics remains unclear. to this aim, we developed a pcr-rflp test that discriminates the five known wolbachia groups found in this host complex. we further examined the wolbachia genetic diversity, the variability in the coinherited host mitochondria and their partitioning among members of the cx. pipiens complex, in order to assess the impact of wolbachia on host population structure.  there was a strong association between wolbachia and mitochondrial haplotypes indicating a stable co-transmission in mosquito populations. despite evidence that members of the cx. pipiens complex are genetically distinct on the basis of nuclear dna, the association of wolbachia and mtdna with members of the cx. pipiens complex were limited. the wolbachia wpip-i group, by far the most common, was associated with divergent cx. pipiens members, including cx. quinquefasciatus, cx. pipiens pipiens form pipiens and cx. pipiens pipiens form molestus. four other wpip groups were also found in mosquito populations and all were shared between diverse cx. pipiens members.  this data overall supports the hypothesis that wpip infections, and their allied mitochondria, are associated with regular transfers between cx. pipiens members rather than specific host associations. overall, this is suggestive of a recent and likely ongoing cytoplasmic introgression through hybridization events across the cx. pipiens complex. endosymbiosiswolbachiacytoplasmic introgressionculex pipiens complex 