FIG. 4.

Subspecies and sex determination of three SIVcpz-infected, nonhabituated chimpanzees from Kalande. (A) Phylogenetic tree of mitochondrial DNA sequences. D-loop sequences (498 bp) amplified from three SIVcpz vRNA-positive fecal specimens (boxed) from Kalande were compared with mtDNA sequences from other eastern chimpanzees (P. t. schweinfurthii). The mtDNA sequence for Ch-No has been reported previously (18). Sequences for Ch-06, Ch-22, Ch-45, Ch-37, Ch-30, and Ch-Ni have been determined in this study. The tree was obtained by maximum likelihood analysis of 377 gap-stripped sites by using the REV + Gamma substitution model implemented in BASEML from the PAML package (PAML, software version 3.0b; University College London, London, United Kingdom) testing 48 tree topologies obtained from NUCML from the MOLPHY package (program package for MOLecular PHYlogenetics, software version 2.2; Institute of Statistical Mathematics, 4-6-7 Minami-Azabu, Minato-ku, Tokyo 106, Japan). Asterisks denote clades to the right found in at least 70% of bootstrapped replicates analyzed by the neighbor-joining method implemented in CLUSTAL W (53) using Kimura's two-parameter correction (27). The three Kalande sequences (boxed) represent different haplotypes within the P. t. schweinfurthii radiation, indicating that the samples came from three different individuals. Ch-30 and Ch-37 (in parentheses) have haplotypes identical to those of Ch-64 and Ch-45, respectively. The tree is rooted on sequences from the subspecies P. t. verus and P. t. vellerosus. (B) Sex determination of Ch-64, Ch-70, and Ch-71 from Kalande. A region of the amelogenin gene, known to contain a 189-bp deletion near the centromere of the Y, but not the X, chromosome, was amplified (16). Fecal DNA from known male (Ch-06, Ch-05, and Ch-16) and female (Ch-09, Ch-25, and Ch-01) chimpanzees from Kasekela was analyzed for control. Sex determination for Ch-64, Ch-70, and Ch-71 was performed in duplicate.