Pangenome Analysis Reveals Gene Content Variation and Evolutionary Dynamics Across the Culex pipiens Species Complex

The Culex pipiens species complex contains the principal northern-hemisphere vectors of West Nile virus, lymphatic filariasis and several arboviruses; yet, the genomic basis of its diversity remains poorly resolved. We present the first pangenome characterization of the four widely recognized Cx. pipiens complex forms ( Cx. pipiens , Cx. pallens , Cx. quinquefasciatus , Cx. molestus ), with Cx. tarsalis as outgroup. From chromosome-scale assemblies (533–790 Mb) and unified annotations transferred from the Cx. quinquefasciatus reference, we identified 16,568 orthogroups. Restricted to the ingroup, 92,821 genes (98.2 %) were partitioned into 11,284 core (71.1 %), 3,726 shell (23.5 %) and 871 cloud (5.5 %) orthogroups. Form-specific cloud orthogroups were unevenly distributed (χ² = 71.9, p = 1.7 × 10⁻¹⁵), with Cx. molestus carrying the largest set. A concatenated 8,067-locus phylogeny placed Cx. pallens and Cx. quinquefasciatus as sisters with full bootstrap support but only 58.9 % gene-tree and 45.1 % site concordance at the deep ingroup node, a pattern consistent with prior evidence that Cx. pallens arose through hybridization between Cx. pipiens and Cx. quinquefasciatus . CAFE5 detected 582 gene families with significant rate departures, and inferred expansions outnumbering contractions on every lineage (counts across all orthogroups with an inferred size change; Cx. quinquefasciatus greatest, 3,733 expanded vs. 729 contracted). Whole-genome ANI ranged 93.1–94.7 % (skani); synteny was conserved at the chromosome scale (74–77 % collinear anchors) with 258–294 inversions ≥ 100 kb per pair. Transposable elements occupied a uniform genome fraction (52.7–54.4 %). These results refine the divergence framework for the complex, provide evidence consistent with a hybrid origin of Cx. pallens , and establish a pangenomic baseline for future population-level resequencing.