Viral molecular diversity of the Nef encoding region in gut tissues of HIV-1 patients. Viral Nef sequences were more diverse (higher mean total distance (d)) for the duodenum and colon compared to the stomach and esophagus (A). Moreover, viral evolution tended towards a more diverse Nef protein in the colorectum as reflected by a significantly higher mean total non-synonomous distance (dN, i.e. amino acid changing mutations) (B). A similar analysis of the RT coding region of HIV-1, also revealed significant differences in viral molecular diversity in the different tissues for both mean total distance (d) (C) and non-synonomous distance (dN) (D). These observations indicated that different selection pressures were acting in different parts of the gut depending on the viral region. (* = p < 0.05, ** = p < 0.01 ***, = p < 0.001, Dunn's multiple comparison test).