New photomap of Anopheles (Nyssorhynchus) darlingi Root, 1926, is described for a population from Guajará-Mirim, State of Rondonia, Brazil. The number of sections in the previous A. darlingi reference map was maintained and new subsections were added to the five chromosome arms. Breakage points of paracentric inversions had been previously incorporated into the photomap of this species. An additional inversion is reported, called 3Lc, totaling 14 inversions in the A. darlingi chromosome arms. The proposed photomap is potentially useful for further evolutionary studies in addition to physical and in silico chromosome mapping using A. darlingi genomic and transcriptome sequences. Furthermore, in our attempt to compare sections of the 2R chromosome arm of A. darlingi with Anopheles funestus, Anopheles stephensi, and Anopheles gambiae, we found great differences in the arrangement of the polytene chromosome bands, which are consistent with the known phylogenetic divergence of these species.
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