LAUSR

Whether the Colobus angolensis that reside in the fragmented forests in eastern Kenya and Tanzania represent one subspecies or two has been debated for 50 years. Morphological and more recent genetic and ecological studies suggest that these populations represent two subspecies, C. a. palliatus and C. a. sharpei. However, their distribution of mitochondrial variation remains unresolved since the genetic study only characterized four populations at the range ends. Therefore, we characterized five populations in the area of the hypothesized subspecies divide. We identified eight new haplotypes which, combined with those previously identified, provided 26 haplotypes from nine populations for analysis. Haplotypes found south of the Rufiji River cluster together but separately from northern haplotypes. The largest sequence differences within cytochrome b occur between population pairs representing opposite sides of the river; their mean difference (1.5%) is more than that of other primate subspecies. Analysis of molecular variance attributes most of the variation to that north versus south of the river. These results support the previous subspecies distinction between C. a. palliatus (northern) and C. a. sharpei (southern), divided by the Rufiji River. The estimated time of the most recent common ancestor of all haplotypes indicates that the subspecies have been isolated from each other for approximately 550,000 years. The common ancestor of northern and southern haplogroups was 370,000 and 290,000 years ago, respectively. Nevertheless, the correlation between genetic and geographic distances suggests that isolation‐by‐distance contributed to population structuring. Significant variation among populations, with only three haplotypes shared between populations, also indicates that an extended period of isolation drove population distinctiveness. Considering these results, we evaluate hypotheses about the founding and differentiation of these subspecies during Pleistocene climatic fluctuations and propose a novel, more direct migration route from Central Africa to their current range navigating Lake Tanganyika, the central Tanzanian corridor, and the Rufiji River.