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 ({chi}{superscript 2} = 71.9, p = 1.7 x 10-{superscript 1}), 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. HighlightsO_LIFirst chromosome-scale pangenome of the Culex pipiens species complex. C_LIO_LI16,568 orthogroups partitioned into core, shell and cloud compartments. C_LIO_LIPhylogenomic discordance supports a hybrid origin for Cx. pallens. C_LIO_LICAFE5 reveals strong, lineage-wide gene-family expansion across the complex. C_LIO_LIConserved synteny and [~]53% transposable-element content across all genomes. C_LI