Overall genetic diversity was similar among populations despite several loci (CxqTri4F, CxqCTG10, Cxpq78) having lower diversity values in some populations (). 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 (HO
), indicating an excess of heterozygotes ().
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 ). 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 (). 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 FST 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 (). Nonsignificant pairwise FST 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 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 (), 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
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
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
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
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.