Master thesis, Christina Hvilsom (KU-BIO), Frands Carlsen (Zoo), Anders Fomsgaard (Statens Serum Institut), Bo Vest Pedersen (KU-BIO)

Chimpanzee (Pan troglodytes) subspecies are classified as endangered in the wild. Subspecies characterization of chimpanzees in captivity is important to help guide conservation breeding programme efforts. Current classification of chimpanzee subspecies has yielded contradictory results. Only in the case of mtDNA has it to some extent been possible to separate the different subspecies. A more efficient method for subspecies determination will help consolidate the existing European breeding programme for Western Chimpanzees (P. t. verus), and improve the management of the remaining chimpanzee population in Europe. Because the CD4 receptor belongs to the immunoglobulin superfamily a variation in this gene could be expected and perhaps provide enough discriminative power to be useful for subspecies determination. The CD4 molecule interacts with MHC-II and is the primary receptor for HIV and SIV. Relatively little is known about the genetic diversity of the chimpanzee CD4 receptor. Only very few (two) sequences are available, and with no certified origin of the subspecies.

The aims of this master thesis were threefold: First, to characterize of the genetic variation of the CD4 gene among the four chimpanzee subspecies P. t. verus, P. t. vellerosus, P. t. troglodytes and P. t. schweinfurthii. Second, to evaluate the ability of CD4 sequencing to distinguish between the chimpanzee subspecies. Third, to evaluate the ability of CD4 sequencing to distinguish between “pure” and hybrid individuals, and if possible the descent of the hybrid individuals. DNA samples were collected in Cameroon, Africa from a total of eight P. t. troglodytes, six P. t. vellerosus and two P. t. schweinfurthii with known origin, ensuring correct accordance between the geographic location of the chimpanzees, and the genetic subspecies determination. Additional six P. t. verus specimens from Copenhagen Zoo, Denmark were included and supplementary nine individuals of unknown or hybrid origin. The CD4 receptor gene was directly sequenced from PCR amplified PBMC DNA and RNA, and subsequently cloned to study nucleotide polymorphisms. Results show that the P. t. verus CD4 sequences are conserved and form an individual monophyletic clade, differing from the P. t. vellerosus, P. t. troglodytes and P. t. schweinfurthii CD4 sequences. The P. t. vellerosus and P. t. troglodytes CD4 sequences are polymorphic compared to CD4 of the P. t. verus subspecies. Individuals previously assumed to be ”pure” P. t. vellerosus, P. t. schweinfurthii and P. t. troglodytes were clearly hybrids according to our method, implying that some level of interbreeding between populations of different subspecies in the subspecies boundary areas is ongoing between P. t. vellerosus and P. t. troglodytes as well as P. t. troglodytes and P. t. schweinfurthii. Through PCR and cloning we were able to distinguish between the pure and mixed chimpanzees in Zoos and determine the descent of the chimpanzees with heterozygous alleles. Future perspectives include testing of more known pure chimpanzee subspecies in order to verify results. Furthermore testing of the remaining European chimpanzee population will consolidate the existing breeding programme for P. t. verus, and improve the management of the entire chimpanzee population.