1. Measuring genetic divergence and diversity in Pennant’s red colobus monkey and Preuss’s red colobus monkey: Implications for the conservation of two endangered taxa Laura Stulken and Dr. Nelson Ting Department of Anthropology, University of Iowa INTRODUCTION Many primate populations across Africa are considered to be endangered, a result of the close contact they have with humans. One of the most endangered species in Africa is the Pennant’s red colobus (Procolobus pennantii). The International Union for Conservation of Nature Red List has declared the species to be critically endangered, based on studies that have shown Pennant’s red colobus populations have declined by 80% in the past 30 years. The main threat to the Bioko red colobus is hunting, and the main threats for the Preuss’s red colobus are hunting and habitat degradation (www.iucnredlist.org). Genetic studies can be useful in the conservation efforts of endangered species. Molecular research can provide information about species’ evolutionary histories and their distinction from other similar lineages. Endangered species or subspecies that can be proved to be genetically divergent or distinct from similar lineages can then receive more conservation attention in order to preserve biodiversity. In addition, genetic studies can give us clues about the health of a population by studying diversity. Populations with reduced genetic diversity are more at risk for illness than those with higher genetic diversity. Small populations with low diversity are especially at risk for inbreeding depression and have less ability to adapt to change. This research looks at the genetics of two different populations of endangered red colobus monkey in West Africa. The Bioko red colobus (Procolobus pennantii pennantii) is endemic to Bioko Island, Equatorial Guinea, off the coast of West Africa (Image 1). Preuss’s red colobus (Procolobus pennantii preussi) is found mainly in Korup National Park in southwest Cameroon (Image 2). Until now, there has been only limited genetic data collected on these two populations of endangered red colobus monkey. The results of this study, as well as those from further studies, can hopefully be used in conservation efforts for the red colobus monkey on Bioko Island and in Korup National Park. METHODS Genetic sequences from both Procolobus pennantii pennantii (Bioko Island) and Procolobus pennantii preussi (Korup National Park) were compared. Samples of P. p. pennantii (n=9) were mainly tissue samples from bushmeat markets. Mainland P. p. preussi samples (n=13) were mainly fecal samples. Mitochondrial DNA was extracted from the samples, and the target regions of cytochrome b and HV1 (~2000 base pairs) in the Control Region of the mitochondrial genome were amplified in these individuals (Image 3). The sequences from this region were aligned with the computer program Geneious. Sequences within and between the two populations were compared and used to create a maximum likelihood tree (Figure 2) using BEAUti, BEAST, and FigTree to show genetic divergence. MEGA 4 was used to compute genetic diversity (Figure 1). RESULTS DISCUSSION These preliminary results are useful to begin looking at how genetic data from these two populations can be used in their conservation. Genetic diversity was found to be lower in the Procolobus pennantii pennantii population than in the Procolobus pennantii preussi population (Figure1). This suggests the island population has gone through a more severe genetic drift event (bottleneck or founder effect) than the population in Korup National Park. In Figure 2 we can see that the Bioko population diversified later than the population in Korup National Park, which may explain why there is less accumulated diversity. The next step in analyzing this diversity data is to compare the within population diversity at this region of the genome of both populations to other primate species and subspecies. This can help us understand how diverse these two population are compared to other primate populations, and how much attention needs to be given to preserving their current genetic variation. P. p. pennantii and P. p. preussi formed different clades on the phylogenetic tree (with the exception of one sample, P. p. preussi K12D). These two populations formed distinct groups from the black and white colobus (Colobus guereza) and the Western red colobus (Procolobus badius). The populations form two distinct groups in the tree, showing they are indeed divergent groups, but they group together when compared to the Western red colobus, showing they are similar taxa, and thus reflecting their evolutionary relationship. This was expected because the island population is thought to have come from this mainland population when the island became separated from mainland Africa. This similarity is reflected by the result that the diversity between the groups is the same as the diversity within one of the populations. There is relatively little difference between the groups. The next step in analyzing this divergence data is to compare group divergence of these two populations to other taxa to determine how distinct these lineages are from others. If the two populations are genetically distinct enough from other similar taxa, they can potentially be raised from a subspecies level to a species level, and they may in turn receive more conservation attention. Continued research in this project will include an expanded dataset for both populations, and the incorporation of microsatellite marker data from the nuclear genome. This microsatellite data will tell us more about the genetic diversity within and between the populations. The results of this continued research can be incorporated into the existing knowledge about the status of the two endangered populations. If the results are significant, they can be used by organizations like the ICUN Red List or NGO’s who can direct more attention to conserving the populations. Hopefully this research can be used to show the need for increased government action, increased forest protection, and increased funding for the forests in Bioko Island and Korup National Park to help conserve these endangered animals. REFERENCES ACKNOWLEDGEMENTS Procolobus pennantii pennantiiProcolobus pennantii preussi Preliminary results about molecular diversity and divergence in the two populations: Diversity Genetic diversity of the two populations of red colobus monkey was found using a pairwise distance matrix in MEGA 4. Figure 1 shows the uncorrected p-distances of the Bioko Island and Korup National Park populations. The between group means were also compared to show the difference between the two populations. Figure 1 - Genetic diversity, using pairwise distance matrix Procolobus pennantii (Bioko Island) 0.004 ± 0.001 (SE) Procolobus preussi (Korup National Park) 0.011 ± 0.001 (SE) Between group means 0.011 ± 0.003 (SE) Divergence A maximum likelihood phylogenetic tree (Figure 2) was created using the software BEAUti, BEAST, and FigTree. Figure 2- Maximum likelihood tree from mtDNA showing molecular divergence between the two populations. Papio hamadryas (Hamadryas baboon) and Theropithecus gelada (Gelada baboon) were chosen as outgroups for comparison (their split is well-understood to be ~5 MYA). Colobus guereza is the black and white colobus, and Procolobus badius is the Western red colobus. Nodes are labeled with posterior probability, and the scale bar represents 2 MY. Image 3- Mitochondrial genome, Cyt b and Control Region found at top (Ting 2008) Image 1- Bioko Island, location of Procolobus pennantii pennantii (Bioko Biodiversity Protection Program) Image 2- Korup National Park, location of Procolobus pennantii preussi (Linder 2008) P. p. pennantii 19 P. p. pennantii 18 P. p. pennantii 47 P. p. pennantii 53 P. p. pennantii 31 P. p. pennantii 23 P. p. pennantii 03 P. p. pennantii 02 P. p. pennantii 01 P. p. preussi K14 P. p. preussi K12C P. p. preussi K11 P. p. preussi K10 P. p. preussi K15 P. p. preussi K19 P. p. preussi K20 P. p. preussi K21 P. p. preussi K16 P. p. preussi X06 P. p. preussi S03 P. p. preussi S04 Procolobus badius Bioko Biodiversity Protection Program, www.bioko.org International Union for Conservation of Nature Red List of Threatened Species, www.iucnredlist.org Linder, J.M. (2008). The impact of hunting on primates in Korup National Park, Cameroon: implications for primate conservation. PhD dissertation, The City University of New York. Ting, N. (2008). Mitochondrial relationships and divergence dates of the African colobines: evidence of Miocene origins for the living colobus monkeys. J. Hum. Evol. 55: 312-325. I would like to thank Dr. Nelson Ting and Joel Corush for all of their help with this project. Thank you also to the Iowa Center for Research by Undergraduates for funding this work. Photographs from Nelson Ting and Joshua Linder. Papio hamadryas Theropithecus gelada Colobus guereza P. p. preussi K12D 1.9136.8091 1 1.9704.4160 1