The Species-Specific Inversion Polymorphism of the X Chromosome in Anopheles messeae and Anopheles daciae Is Based on the Common Ancestral Variant X1

<b>Background/Objectives</b>: Chromosomal inversions play an important role in the evolution of insects by forming genetic barriers between closely related species and facilitating local adaptation. Polymorphic inversions in malaria mosquitoes of the Maculipennis subgroup have been studied for over 50 years, yet the evolutionary ancestry of the gene orders remains unknown. In this study, we mapped the genes flanking the breakpoints of two polymorphic X-chromosome inversions in the cryptic species <i>Anopheles messeae</i> and <i>Anopheles daciae</i> of the Maculipennis subgroup. <b>Methods</b>: We used an iterative mapping approach to define the breakpoint regions, selecting flanking markers based on the genome assembly of the reference species, <i>Anopheles atroparvus</i>. To identify the ancestral X chromosomal arrangement in <i>An. messeae</i> and <i>An. daciae</i>, we developed and implemented the genomic inversion calculator (GIC), which uses greedy heuristics to determine the shortest evolutionary scenario of rearrangements. <b>Results</b>: Our knowledge of the relative genomic positions of the inversion breakpoints in <i>An. daciae</i> and <i>An. messeae</i> enabled us to use the <i>An. atroparvus</i> genome as an outgroup and the GIC tool to show that the X0 and X2 arrangements emerged independently along the evolutionary lineages of <i>An. daciae</i> and <i>An. messeae</i>, respectively, based on the X1 arrangement. <b>Conclusions</b>: These results refine the structure and boundaries of the X chromosome rearrangements and reconstruct the sequence of evolutionary events in the cryptic complex <i>An. messeae</i>-<i>An. daciae</i>, demonstrating that the X1 arrangement is ancestral. This study lays the groundwork for analyzing the molecular organization of breakpoints, the mechanisms of inversion formation, and their role in speciation.