The Culex pipiens complex is an important group of mosquito vectors that transmit several encephalitic viruses, including West Nile and St. Louis encephalitis viruses.1,2 Members are found in temperate and tropical regions throughout the world, and the complex is notable for the variety of ecological, behavioral, and physiological adaptations present within such a closely related group of organisms. In the United States, members include Cx. pipiens form pipiens L., Cx. quinquefasciatus Say, their hybrids, and the autogenous form of Cx. pipiens known as form molestus Forskäl. Adaptations among taxa in the complex include epidemiologically relevant characters such as the presence or absence of seasonal reproductive diapause, which can influence the degree and nature of virus transmission,3 and whether females are autogenous (capable of producing a first batch of eggs without a blood meal). Variation in vector competence has been observed in Cx. pipiens complex populations in the state of California, and it has been suggested that such variation has a genetic component.4–6

A characteristic that potentially effects the distribution of adaptations within the Cx. pipiens complex is that Cx. pipiens and Cx. quinquefasciatus are fully interfertile, as are the resultant hybrids. This feature has resulted in a large stable hybrid zone across the United States, the limits of which Barr7 set at 36°N and 39°N based on measurements of the dorsal and ventral arms of the male genitalia (DV/D ratio). Subsequent work using microsatellites8–11 has indicated that the hybrid zone extends farther north and south than suggested by Barr.7 Whether this difference is caused by hybrid zone expansion over time or to the improved power of microsatellites over morphology to detect admixed mosquitoes is unknown, and both scenarios may be true. Regardless, there are areas in the United States where Cx. pipiens complex populations consist largely of genetically admixed mosquitoes. Interspecific hybridization produces new genotypes through genetic recombination, and thus provides natural selection with more variation on which to act. Such genetic variation may enable populations to adapt to local conditions, and can lead to genetic differentiation. Recent work has examined the possible effect of genetic introgression on host choice among Cx. pipiens complex mosquitoes, and a small number of studies have suggested the possibility that genes that confer a preference for biting humans could be transferred from form molestus to form pipiens mosquitoes, which could potentially increase the transmission of arboviruses to humans.12–15

The interaction of these two features of the complex, interfertility and a variety of life-history strategies makes the taxonomic designation of a particular Cx. pipiens complex population occasionally ambiguous. Characterizing the Cx. pipiens complex in California is made more challenging because its distribution is also influenced by geographic (and associated temperature) regimens that are more complex than simple clines. Barr7 examined DV/D ratios from individuals in several populations in California when describing the distribution of species and hybrids in North America, and noted the existence of admixed populations. A dissertation by Iltis (Iltis WG, unpublished dissertation) concluded that Cx. quinquefasciatus was found in expected locations in the southern part of the state, but also north of Sacramento, whereas Cx. pipiens was found in Sacramento and again in the northern part of the state. The work of Iltis also characterized hybridization within the complex, noting several stable zones of hybridization. Tabachnick and Powell16 sought to confirm the results of the study of Iltis, and in addition to DV/D ratios, examined several allozyme loci. Although morphologic data from both studies agreed with respect to the distribution of species (then called subspecies) around Sacramento, variation in allozyme frequencies did not correlate well with that of DV/D ratios. Tabachnik and Powell16 also made the distinction between hybrid populations made up of largely F₁ individuals and the stable freely interbreeding populations they observed, and noted that taxonomic classification of populations in California as Cx. pipiens or Cx. quinquefasciatus for epidemiologic reasons could be misleading.

Urbanelli and others 17 used DV/D ratios and a panel of six allozymes that included several new loci to assay populations in California. They noted that temperature alone was insufficient to explain the distribution of DV/D ratios and that the pattern of allozyme frequencies suggested that populations were responding to microclimatic changes over the past several decades. This response resulted in the movement of Cx. pipiens southward, which shifted the southern limit of the hybrid zone. Comparing Cx. pipiens complex populations in California and South Africa using DV/D ratios and 11 isoenzyme loci, Cornell and others18 noted differences between the two sampling locations, which suggested Cx. pipiens complex taxa were less taxonomically distinct in California than in South Africa, where populations displayed limited to no introgression between species. Using a single-gene assay (Ace.2) and DV/D ratios to identify Cx. pipiens complex mosquitoes in Fresno County, California (located within the hybrid zone) McAbee and others19 sought to correlate West Nile virus infection rate with taxonomic designation. They found both identification methods uninformative with regard to classifying Cx. pipiens complex individuals and concluded that there is “no appropriate diagnostic character” to separate Cx. pipiens from Cx. quinquefasciatus in California.

The presence of autogenous individuals in California was noted by Barr.7 Iltis also mentions autogeny in CA populations, but because the DV/D ratio is identical in form molestus and form pipiens, few morphologic studies have knowingly included form molestus mosquitoes. A recent study by Strickman and Fonseca20 used morphology and eight microsatellite loci to characterize autogenous individuals in 13 above-ground sites around San Francisco, California. They observed a variety of phenotypes among and within sites, and suggested such variety could promote adaptation to local microclimates.

As the above mentioned studies suggest, there is considerable ambiguity in the taxonomic designations of Cx. pipiens complex populations in California. Furthermore, to our knowledge, the degree and extent of genetic admixture of Cx. pipiens complex populations in the state has not been recently characterized using a north–south transect. Although it is true that no one characteristic reliably distinguishes taxa within admixed populations, a panel of microsatellite markers can be useful for this purpose. Our aim in the current study was to characterize populations that represent much of the genotypic variation in California with microsatellites. Specifically, our objectives were to 1) quantify genetic diversity and differentiation within and among populations; 2) determine the most likely number of genetically distinct clusters, and to assign individuals to those clusters; 3) evaluate the respective contributions of Cx. pipiens and Cx. quinquefasciatus to admixed clusters; 4) determine whether other Culex taxa have introgressed into Cx. pipiens complex populations in California; and 5) determine whether morphological determinations of autogeny (or anautogeny) are consistent with genetic cluster assignments.

Overall genetic diversity was similar among populations despite several loci (CxqTri4F, CxqCTG10, Cxpq78) having lower diversity values in some populations (Table 2). Most of the significant departures from HWE were caused by an excess of homozygotes (i.e., a deficit of heterozygotes). Often, an excess of homozygotes is suggestive of the Wahlund Effect,44 in which allele frequencies are sampled after some degree of population subdivision has occurred. However, in the Manhole Sacramento and Zoo populations, there was a small number of significant HWE departures in the opposite direction (H_O > H_E), indicating an excess of heterozygotes (Table 2).

These findings were consistent with genetic characterizations of populations whose allele frequencies have changed because of some perturbation.45 One possibility is that the Manhole Sacramento and Zoo populations have each experienced a genetic bottleneck. Another possibility, although not mutually exclusive, is that the Manhole Sacramento and Zoo populations were receiving gene flow from genetically dissimilar populations, a condition referred to as isolate breaking,44 which is supported by the high degree of LD (27 in Manhole Sacramento and 19 in Zoo) in both populations. The Bottleneck analysis suggested both the Manhole Sacramento and Zoo populations have experienced bottlenecks, but also detected the signature of a bottleneck in several other populations that would not be expected to have them. A recent review by Peery and others46 suggests that the program Bottleneck can give spurious results when sample sizes are small, and that results can be affected by the duration and time since the bottleneck event. Conversely, some mosquito populations regularly experience reductions in numbers because of vector control efforts, which could influence allele frequencies in these natural populations even though they are in HWE. For example, Cartaxo and others47 found that genetic diversity of populations of Cx. quinquefasciatus in Brazil decreased over a three-year period because of control efforts. Our study used specimens collected by vector control programs from sites known to harbor mosquitoes and probably included in control programs. Nevertheless, the Manhole Sacramento and Zoo populations, and to a lesser degree Manhole Old Sacramento, had distinct patterns of HWE and LD.

Another commonality of the Zoo and Manhole Sacramento populations was that both had autogenous mosquitoes. The low genetic diversity found in the Manhole Sacramento population was in contrast with the Zoo population's genetic diversity values, which were towards the middle of the distribution of values. One explanation could be that the two populations have had different patterns of gene flow subsequent to the proposed genetic bottleneck, in which Manhole Sacramento has been isolated and the Zoo has been receiving gene flow. Several findings support this assertion. First, Structure assigned many Zoo specimens to Cluster M when the Washington and Colorado Cx. pipiens populations were included, but these individuals were assigned to Cluster P when they were excluded (compare panels A and B in Figure 3). Also, despite the presence of large proportions of autogenous mosquitoes at the Zoo site, the neighbor-joining tree placed this population away from the other autogenous populations (Figure 2). Finally, of the 35 specimens examined morphologically and determined to be autogenous in the Zoo population, most (71%) assigned to Cluster M, suggesting at least some individuals are genetically distinct enough to be classified as form molestus. Taken together, it appears that the allele frequencies in the Zoo population were less fixed than in the Manhole Sacramento population. This finding might have been caused by characteristics of the Zoo collection site, which made it more accessible to colonizing mosquitoes. The Zoo site was a catch basin containing water 1–1.5 meters below street level and thus was an enclosed underground site, although not as isolated as the Manhole Sacramento site (Nelms B, unpublished data).

The AMOVA results indicated that although most diversity resides at the level of the individual, almost twice as much genetic variation was partitioned among populations versus among individuals within populations. This distribution of variance was similar to that in another study48 we conducted that involved form molestus mosquitoes in Chicago, Illinois, and New York, New York, but contrasted with our work with Cx. quinquefasciatus, Cx. pipiens, and their hybrids along a transect from New Orleans, Louisiana, to Chicago, Illinois, in which variation was distributed equally at the two upper levels of organization in the AMOVA.8 Inclusion of a group with low genetic diversity such as form molestus might result in a greater proportion of variance at the among-population level.

The overall F_ST value reflects differences between many of the pairs of populations in this study, as well as among the four genetically distinct groups discerned by the Structure analyses. With regard to comparisons among pairs of populations, the largest degree of genetic differentiation was between the Cx. quinquefasciatus populations and the autogenous populations, followed by that between Cx. quinquefasciatus populations and Cx. pipiens populations (Table 4). Nonsignificant pairwise F_ST values resulted from comparisons between pairs of populations in the areas surrounding Sacramento, suggesting that these populations had a similar mixture of genotypes. The neighbor-joining tree was consistent with these findings, because the populations around Sacramento had low bootstrap support but were located together between the Cx. quinquefasciatus populations, the Cx. pipiens populations, and the form molestus populations. The low bootstrap support might have been caused by the populations being similar mixtures of the other three larger genetic groupings, such that their consensus order on the branch is equivalent to several other possible orders.

If Cx. pipiens form pipiens in the United States can be defined as genetically similar to populations in Washington and Colorado, the Structure results suggest there is a low frequency of Cx. pipiens genotypes in California. When only California populations were included in the analysis, K = 3 was the most likely number of clusters. The addition of Washington and Colorado Cx. pipiens populations resulted in a fourth genetic cluster being derived from the data. The comparison of panels A and B in Figure 3 shows that Cluster X when K = 4 is still present when K = 3. When K = 4, Cx. pipiens individuals represented mostly by the Washington and Colorado populations, form a distinct group. This finding might be caused by the small number of pure Cx. pipiens form pipiens in California, and those individuals although similar enough to group with Cluster X when K = 3, were more similar to the Washington and Colorado Cx. pipiens when those data were included in the analysis. It is clear that mosquitoes in Cluster X are Cx. pipiens complex mosquitoes because the microsatellite loci used in the study amplified in these populations similarly to the other populations and had no unique alleles.

The CLUMPP analyses of the Structure runs with and without the Washington and Colorado populations yielded interesting results that likely reflect the ability of this software to detect genetic structure at more than one level of biological organization. When only California populations were considered, individuals were most consistently assigned to the same cluster when K = 2 or K = 4. When K = 2, genotypes are organized rather broadly into those that belong to Cluster Q and those that do not, and the populations from Turlock northwards appear as one group. The results of the ΔK analysis may have detected only this broad pattern in the data. When K = 4, the analysis appears to detect the small number of Cx. pipiens form pipiens in the northern CA populations, as well as the large number of Cluster X mosquitoes. When the Washington and Colorado populations are included, several stable configurations of cluster assignments were evident: K = 2, K = 3 and K = 5. At K = 3, mosquitoes are still partitioned into logical groups: Cluster Q, Cluster P, and a group comprised of Cluster X and Cluster M. When K = 5, Cluster Q, Cluster P, and Cluster X are present. However, many individuals that assigned to Cluster M when K = 4 were deemed hybrids (i.e., individual specimens did not assign to any cluster with a q value ≥ 0.80). The inability of the program to assign such a large proportion of mosquitoes to a cluster indicated that K = 4 (Clusters Q, P, X, and M) was realistic, despite not having as strong support in the CLUMPP analysis.

Interestingly, removing hybrids (when K = 4) from the subsequent Structure analysis suggests additional population subdivision within Cluster M that was otherwise obscured. In particular, the Manhole Sacramento and Zoo populations are genetically distinct from each other and from the other California populations studied. This finding is consistent with those of previous studies,48,49 which suggested form molestus populations in the United States are genetically divergent from each other and from local above-ground form pipiens populations.

The choice of a q cutoff value of 0.80 may have overestimated the number of pure Cx. pipiens individuals which is somewhat surprising given the low proportions of such mosquitoes in California. Populations that had Cluster P individuals were found as far south as Elk Grove (2%), which is relatively close to the 39°N latitude distribution boundary of Cx. pipiens in the United States reported by Barr.7 However, the two populations above that latitude, Lake and Shasta, had only 3% and 35% of individuals respectively, assigned to Cluster P and < 10% of individuals from the remaining California populations assigned to this cluster. Most genotypes found at the Lake and Shasta sites were not assigned to any cluster because their q values were divided among two or more clusters, indicating that these populations have a high proportion of hybrid individuals. Our findings that Cx. pipiens form pipiens is rare in northern California is consistent with those of a related study on overwintering Cx. pipiens complex females from the Shasta population, where an unexpectedly high proportion of females did not enter reproductive diapause under midwinter conditions.50 Benton, Washington, also had a small proportion (2%) of hybrids, but consisted mostly (98%) of Cx. pipiens form pipiens mosquitoes.

From its position on the DAPC plot (Figure 5), Cluster X appears to be made up of contributions from Cx. pipiens form pipiens, Cx. pipiens form molestus, and Cx. quinquefasciatus. It appears from the location of Cluster X on the plot that its ancestry contains somewhat more form pipiens and form molestus than Cx. quinquefasciatus. If Cluster X was a hybrid only of Cx. pipiens form pipiens and Cx. quinquefasciatus, it would be expected to be located directly between those clouds of points on the DAPC plot. Instead, Cluster X was positioned in the middle of the plot, but clearly was closer to the P and M clusters. In addition, our analyses indicate autogenous mosquitoes in California largely belong to Cluster M, and were more closely related to Cx. pipiens and Cluster X mosquitoes than to Cx. quinquefasciatus. This finding is in contrast to a recent study of Cx. pipiens complex mosquitoes in the San Francisco area by Strickman and Fonseca, which concluded that autogenous mosquitoes there had hybridized with Cx. quinquefasciatus.20 The results of the Loading Plot analysis showed that data from one marker in the microsatellite panel, Cxpq78, was best at discriminating among the groups in the DAPC analysis. For this reason, this marker could be useful in future examinations of introgression in the Cx. pipiens complex.

Several studies have noted the presence of what have been referred to as stable hybrid populations in and around the central valley of California, which historically were presumed to consist of Cx. pipiens–Cx. quinquefasciatus hybrids.16,19 Unlike some areas subjected to temperature extremes that shift the balance of taxa over the course of a year, the existence of areas with entirely admixed populations has been known for some time.16 Our study detected a distinct genetic group (Cluster X) that is closely related to other members of the Cx. pipiens complex, and comprised of Cx. pipiens form pipiens, Cx. pipiens form molestus, and Cx. quinquefasciatus. That the Structure analyses found Cluster X to be a discernible group suggests that it does not consist of F₁ hybrids, which would be the case if there was a high degree of gene flow from the parental taxa. Instead, Cluster X appears to consist of advanced-generation hybrids. An intriguing possibility is that this group may be undergoing speciation as a result of genetic differentiation from the other taxa over time.

Evaluating temporal and spatial patterns of hybridization in California Cx. pipiens complex populations is important to ongoing disease modeling and vector control efforts because the high degree of interfertility within the complex means that advantageous traits can spread via gene flow. For example, McAbee and others19 noted that the same molecular pathways for two kinds of insecticide resistance were present in Cx. pipiens and Cx. quinquefasciatus populations from California. Although the two mechanisms might have evolved in each taxon independently, the authors emphasize a more likely scenario in which these advantageous mutations arose in one taxa and spread to the other via interspecific hybridization. Another example, based on more recent work, involves a shift in host preference in Cx. pipiens form pipiens. Several studies12–15 have suggested gene flow to form pipiens from form molestus is a contributing factor to comparatively high rates of feeding on humans in populations of Cx. pipiens in the midwestern and eastern United States. Although form molestus is believed to have evolved as an urban mosquito in close proximity to humans, form pipiens is believed to prefer feeding on birds. Interestingly, recent blood meal analysis studies in southern51 and central52,53 California indicate that few Culex pipiens complex females feed on humans, even in urban Los Angeles or Sacramento. If traits such as insecticide resistance and host preference can transfer among closely related forms, other traits that are genetically controlled could also transfer, such as those related to vector competence, autogeny, and seasonal diapause (or the absence of it). The current study is a snapshot of gene flow within the Cx. pipiens complex in California. Subsequent sampling and genetic analysis of these populations would enable inferences to be made as to how the taxonomic composition of populations is changing over time.

Given the high degree of admixture in this system, it was logical to assess whether related species of Culex have contributed genes to the California Cx. pipiens complex populations in the current study. The Structure analysis that included Cx. pipiens pallens mosquitoes from Japan clearly showed that taxon to be distinct from the California populations. The two other Culex taxa whose DNA was assessed with the panel of microsatellites, Cx. stigmatosoma and Cx. restuans, proved not to be similar genetically to the Cx. pipiens complex and most of the microsatellite loci did not amplify.

Comparing the data from morphologic assessments of autogeny with those from the cluster assignments in Structure indicate a fairly good ability of this panel of microsatellites to detect autogenous mosquitoes. In the Zoo population, most (71%) individuals that were morphologically autogenous and assayed with the panel of microsatellites were correctly assigned to Cluster M. The percentage of correct determinations was lower for the other two populations examined, with Manhole Old Sacramento having 44% of autogenous individuals assigned to Cluster M and Dave B House, where one of eight individuals was assigned to Cluster M. The remaining morphologically autogenous individuals in these populations were either assigned to Cluster X or were deemed hybrids because of low q values. None of the morphologically autogenous specimens was assigned to the P or Q clusters. The variety of genotypes present in morphologically autogenous mosquitoes suggests specimens with diverse genetic backgrounds may express autogeny. Given the high proportion of hybrid individuals found especially in populations around Sacramento, it is unknown whether a similar diversity of genotypes will persist over time, or whether subsequent sampling would find ever larger proportions of morphologically autogenous mosquitoes that would be assigned to Cluster M.

The diversity of epidemiologically important life history traits that vary within the Cx. pipiens complex combined with extensive hybridization and adaptation to a variety of local conditions make it an important group of vector species to study. Our study measured genetic diversity and differentiation among California Cx. pipiens complex populations, and characterized the degree and extent of hybridization within the state. Our data will add to future work on Cx. pipiens complex populations in California and elucidate whether the observed patterns of genetic variation continue to evolve, or represent a stable configuration of genotypes.