A New Chromosomal Phylogeny Supports the Repeated Origin of Vectorial Capacity in Malaria Mosquitoes of the <em>Anopheles gambiae</em> Complex

<div><p>Understanding phylogenetic relationships within species complexes of disease vectors is crucial for identifying genomic changes associated with the evolution of epidemiologically important traits. However, the high degree of genetic similarity among sibling species confounds the ability to determine phylogenetic relationships using molecular markers. The goal of this study was to infer the ancestral–descendant relationships among malaria vectors and nonvectors of the <em>Anopheles gambiae</em> species complex by analyzing breakpoints of fixed chromosomal inversions in ingroup and several outgroup species. We identified genes at breakpoints of fixed overlapping chromosomal inversions 2Ro and 2Rp of <em>An. merus</em> using fluorescence <em>in situ</em> hybridization, a whole-genome mate-paired sequencing, and clone sequencing. We also mapped breakpoints of a chromosomal inversion 2La (common to <em>An. merus</em>, <em>An. gambiae</em>, and <em>An. arabiensis</em>) in outgroup species using a bioinformatics approach. We demonstrated that the “standard” 2R+^p arrangement and “inverted” 2Ro and 2La arrangements are present in outgroup species <em>Anopheles stephensi</em>, <em>Aedes aegypti</em>, and <em>Culex quinquefasciatus</em>. The data indicate that the ancestral species of the <em>An. gambiae</em> complex had the 2Ro, 2R+^p, and 2La chromosomal arrangements. The “inverted” 2Ro arrangement uniquely characterizes a malaria vector <em>An. merus</em> as the basal species in the complex. The rooted chromosomal phylogeny implies that <em>An. merus</em> acquired the 2Rp inversion and that its sister species <em>An. gambiae</em> acquired the 2R+^o inversion from the ancestral species. The karyotype of nonvectors <em>An. quadriannulatus</em> A and B was derived from the karyotype of the major malaria vector <em>An. gambiae</em>. We conclude that the ability to effectively transmit human malaria had originated repeatedly in the complex. Our findings also suggest that saltwater tolerance originated first in <em>An. merus</em> and then independently in <em>An. melas</em>. The new chromosomal phylogeny will facilitate identifying the association of evolutionary genomic changes with epidemiologically important phenotypes.</p>