Int J Primatol (2012) 33:479–488 DOI 10.1007/s10764-012-9591-6
Genetic Sampling of Unhabituated Chimpanzees (Pan troglodytes schweinfurthii) in Gishwati Forest Reserve, an Isolated Forest Fragment in Western Rwanda Rebecca L. Chancellor & Kevin Langergraber & Sergio Ramirez & Aaron S. Rundus & Linda Vigilant
Received: 16 August 2011 / Accepted: 11 January 2012 / Published online: 17 March 2012 # Springer Science+Business Media, LLC 2012
Abstract Many primate populations currently live in forest fragments. These populations are often unhabituated, elusive, and contain few individuals, making them difficult to study through direct observation. Noninvasive genetic methods are useful for surveying these unhabituated populations to infer the number and sex of individuals and the genetic diversity of the population. We conducted genetic analysis on 70 fecal samples from eastern chimpanzees (Pan troglodytes schweinfurthii) in Gishwati Forest Reserve, a forest fragment in western Rwanda. We genotyped all but two of these samples using 12 autosomal and 13 Y-chromosome microsatellite markers previously used in analyses of other chimpanzee populations. The genetic data show that these samples represent a minimum of 19 individuals (7 females, 12 males). However, because we may not have sampled all individuals in the population, we also performed mark-recapture analysis with the genetic data and found that the entire
Electronic supplementary material The online version of this article (doi:10.1007/s10764-012-9591-6) contains supplementary material, which is available to authorized users. R. L. Chancellor : A. S. Rundus Great Ape Trust, 4200 SE 44th Avenue, Des Moines, IA 50320, USA
R. L. Chancellor (*) Department of Anthropology, University of California, Davis, Davis, CA 95616, USA e-mail:
[email protected] K. Langergraber : S. Ramirez : L. Vigilant Primatology Department, Max Planck Institute for Evolutionary Anthropology, 04103 Leipzig, Germany K. Langergraber Department of Anthropology, Boston University, Boston, MA 02215, USA A. S. Rundus Department of Psychology, West Chester University, West Chester, PA 19383, USA
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population likely numbers between 19 and 29 individuals. These results are consistent with opportunistic observations of at least 19 individual chimpanzees. Levels of variation at the Y-chromosome microsatellites were similar to those observed in other chimpanzee communities, suggesting that the chimpanzees in this forest are members of a single community. These results provide a baseline count of the number of male and female chimpanzees in the Gishwati Forest Reserve, and the data provide the potential for follow-up studies aimed at tracking individuals over time, thus aiding conservation management of this unhabituated population. Keywords Genotyping . Mark recapture . Noninvasive sampling . Pan troglodytes schweinfurthii . Y-chromosome
Introduction Many primate populations now live in fragmented habitats (Anderson et al. 2007; Chapman et al. 2007; Miller et al. 2004; Pope 1996). Although studying primates in fragments is important for conservation and management of these populations (Baranga et al. 2009; Gibbons and Harcourt 2009; Onderdonk and Chapman 2000), individuals in fragmented populations of primates are often wary and highly mobile, making them difficult to study. In addition, habituation may not be desirable for populations experiencing hunting pressure in the absence of effective law enforcement. Wild apes offer a particular challenge because they typically take years to habituate to human observation (Blom et al. 2004; Doran-Sheehy et al. 2007; Tutin and Fernandez 1991; Williamson and Feistner 2003). Indirect methods such as noninvasive genetic sampling represent viable alternative or complementary approaches to the direct study of animal populations (Kohn and Wagne 1997; Schwartz et al. 2007). One of the most fundamental aspects of a population is its size. Genetic mark-recapture population size estimates can often be more precise than inferences made from direct signs such as feeding remains or nest counts in many species such as otters (Lutra lutra: Arrendal et al. 2007; Hajkova et al. 2009), giant pandas (Ailuropoda melanoleuca: Zhan et al. 2006), gorillas (Gorilla gorilla gorilla: Arandjelovic et al. 2010), and chimpanzees (Pan troglodytes troglodytes: Arandjelovic et al. 2011). In addition to knowing the size of a population, it is also important to understand how the individuals are distributed in social groups, as this will affect the ability of individuals to disperse and ultimately influence the distribution of genetic variation in the population (Archie et al. 2005; Goldberg and Wrangham 1997; Taberlet et al. 1997). The most acute threats to the survival of small isolated animal populations are habitat loss, hunting, and disease (Cowlishaw 1999; Cullen et al. 2000; Goldberg et al. 2008; Oates 1996). However, it has also been recognized that the erosion of genetic diversity can pose a threat to the long-term survival of small populations even in the absence of other threats (Frankham 2005; Johnson et al. 2010; Lande and Barrowclough 1987). One effective way of assessing the genetic diversity of a small population is by comparison with information from other extant populations with varied demographic characteristics to assess whether the study population displays notably less variation (Bergl et al. 2008).
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The Gishwati Forest Reserve, a montane rain forest fragment in western Rwanda, contains a small, isolated, and little-studied population of eastern chimpanzees (Pan troglodytes schweinfurthii). Recent surveys based on counting the nests that chimpanzees make afresh every evening suggested that this population consists of ca. 9–21 individuals (Barakabuye et al. 2007; Chancellor et al. 2012). Here we analyzed DNA from chimpanzee fecal samples collected below nests, along chimpanzee trails, and where chimpanzees had recently been seen or heard over a 9-mo period at Gishwati. The number of different autosomal genotypes obtained represents the minimum number of individuals in the population, and we further determined the sex of these individuals by examining a sex-specific autosomal locus. Using the frequency with which each individual was sampled, we employed mark-recapture analysis to estimate the total population size. In addition, we used Y-chromosome microsatellite analysis to estimate the level of genetic diversity present in the population relative to that observed in other communities of eastern chimpanzees. We focused on comparative analysis of the Y-chromosome because as a uniparentally inherited marker its smaller effective population size should show the impact of reduced population size relatively quickly compared to biparentally inherited autosomal loci (van Oven et al. 2011). In addition, unlike most mammalian species, chimpanzees are male-philopatric and only the females disperse from their natal communities. As a consequence, the members of different communities typically have different Y-chromosome haplotypes. Analysis of variation on the Y-chromosome can thus be particularly useful for determining the number of communities to which a given set of individuals belong (Arandjelovic et al. 2011; Boesch et al. 2007; Langergraber et al. 2007a).
Methods Study Site The Gishwati Forest Reserve is located just south of Volcanoes National Park in western Rwanda (1°49′S, 29°22′E). Since the mid-1970s, Gishwati has been reduced from 280 km2 to its current size of 9 km2 by a series of human activities, including a 200-km2 conversion to an integrated forestry and livestock project supported by the World Bank, a 30-km2 conversion to a military zone, and conversion to camps for displaced persons after the 1994 genocide (Plumptre et al. 2001). This has left the Gishwati chimpanzee population isolated from other chimpanzees by ca. 70 km. The elevation of the reserve ranges from 2020 to 2500 m. The total annual rainfall is 1884 mm, distributed seasonally with a major dry period between June and August (dry season mean040.6 mm). The mean daily minimum and maximum temperature is 15.7 °C and 24.2 °C, respectively (July 2009–June 2010). Focal Subjects Based on opportunistic observations and identifications of individual chimpanzees from November 2008 to February 2010 (209 contact hours), we estimated the Gishwati chimpanzee population to contain 19 individuals, including 6 adult males, 5 adult females, 1 adolescent male, 2 adolescent females, 1 juvenile male, and 4 infants
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(Chancellor et al. 2012). There are at least three other primate species that live sympatrically with the chimpanzees, including l’Hoest’s monkeys (Cercopithecus l’hoesti), golden monkeys (C. mitis kandti), and bushbabies (Galago sp.). Data Collection We collected fecal samples estimated to be ca. ≤24 h old between June 2009 and February 2010 (N070 samples). We collected samples on 20 different days consisting of 5 d between June and August 2009, 10 d in November and December of 2009, and 5 d during February 2010. We collected fecal samples mainly under nests after the chimpanzees had vacated them. We located the nests by following fresh chimpanzee trails using traditional tracking methods. We also collected fecal samples during several systematic “sweeps,” in which the forest was divided into two to three sections and searched simultaneously by several teams. During collection, we placed ca. 5 g of fresh feces into 50-ml collection tubes containing ca. 30 ml of 97 % ethanol. The next day, we transferred the feces into fresh silica tubes for further desiccation and stored them at 4 °C in the field and subsequently at 4 °C in the laboratory (Nsubuga et al. 2004). Laboratory and Data Analysis We extracted DNA using the Qiagen Stool DNA extraction kit and genotyped extracts at 12 autosomal microsatellite loci previously used in studies of other eastern chimpanzee populations (Langergraber et al. 2007b) using the two-step multiplex polymerase chain reaction detailed in Arandjelovic et al. (2009). The primer sequences and annealing temperatures for the 12 loci (D1s1622, D1s1656, D2s1326, D2s1329, D3s3038, D4s1627, D5s1457, D5s1470, D6s1056, D7s817, D10s676, D11s2002) are as previously described (Arandjelovic et al. 2009). To determine the sex of individuals we analyzed all DNA extracts at a sex-specific portion of the amelogenin locus as previously described (Bradley et al. 2001). Two of the samples failed to provide amplifiable DNA. Because we expected to find that some individuals had been sampled multiple times, we conducted an initial round of genotyping by amplifying each locus in triplicate using each of the 68 usable extracts. We then compiled these genotypes into consensus genotypes representing individuals with the aid of Cervus 3.0 and the “identity check” function. In this final set of consensus genotypes, we observed each allele at a heterozygous genotype at least twice, and we observed homozygous genotypes five or more times. We also genotyped all males at 13 Y-chromosome microsatellite loci (DYs439, DYs502, DYs520, DYs533, DYs562, DYs510, DYs517, DYs612, DYs630, DYs469, DYs392, DYs632, DYs588) as previously detailed in our other work on eastern chimpanzees (Langergraber et al. 2007a, b). We used ARLEQUIN (Excoffier et al. 2005) to perform gene diversity (Nei 1987) calculations with the Y data. Mark-recapture analyses use the frequency with which individuals are “recaptured,” i.e., resampled, to derive an estimate of the size of the total population from which the individuals were sampled. We sampled some individuals more than once on the same day, and we did not consider such additional same-day captures in the analysis, as they do not represent true recaptures. We used a maximum-likelihood method implemented
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in Capwire (http://www.cnrhome.uidaho.edu/lecg) to obtain a population estimate. Given the variation among individuals in the number of times they were sampled (see Results), we employed the two innate rates model (TIRM), which assumes heterogeneous capture probability among individuals in a population and assigns individuals as having either a low or high probability of capture (Miller et al. 2005).
Results Our analysis produced 19 unique autosomal genotypes, indicating the presence of ≥19 individuals in this population. These 19 genotypes were 92 % complete over the 12 microsatellite loci. While the two least-complete genotypes were complete only at six loci each, they were clearly distinguishable from the other genotypes (electronic supplementary material [ESM] Table SI). The 19 individuals represent 12 males and 7 females according to the results of the molecular sexing analysis. We sampled the 19 different individuals from 1 to 7 times each, with a mean of 3.11 captures per individual (59 captures/19 individuals). We sampled males more often than females, although the difference was not significant (mean03.75 vs. 2.1 times, P00.0876, unpaired t-test) (Fig. 1). The mark-recapture analysis gave a point estimate of 22 and a 95 % confidence interval of 19–29 individuals. The 12 sampled males in the population had three different Y-chromosome microsatellite haplotypes, which were shared by five, seven, and one male, respectively (ESM Table SII). Consistent with patterns of Y-chromosome variation in other eastern chimpanzee communities, the three haplotypes were extremely similar to one another, differing by a maximum of two mutational steps. The Gishwati chimpanzees have a level of haplotype diversity similar to that of other eastern chimpanzee communities (Table I).
Fig. 1 Number of times we sampled individual males and females.
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Table I Y-chromosome diversity indices in multiple eastern chimpanzee communities Community
No. males typed No. haplotypes No. polymorphic loci Haplotype diversity SD
Gishwati
12
3
2
0.62
0.09
Kanyawara (Kibale) 10
3
4
0.62
0.14
Ngogo (Kibale)
41
8
4
0.56
0.09
Mugiri (Semliki)
6
3
4
0.60
0.22
Sonso (Budongo)
16
4
4
0.74
.06
Data for Kanyawara, Ngogo, Mugiri and Sonso taken from Langergraber et al. (2007a)
Discussion Genetic analysis revealed that there are ≥19 individuals in the Gishwati chimpanzee population, including 7 females and 12 males. Although these individuals were sampled an average of three times each, it is not necessarily the case that we obtained samples from all chimpanzees living in the forest. Therefore, we applied a mark-recapture estimator and obtained an estimated population size of between 19 and 29 individuals. These results correspond well with the 19 individuals we identified opportunistically during behavioral observations (Chancellor et al. 2012). However, it is not necessarily the case that the 19 genetically identified individuals correspond exactly with the 19 observationally identified individuals. Differences may arise because it is notoriously difficult to obtain samples from dependent infants (Boesch et al. 2006), and conversely, genetic samples may have been obtained from adult individuals that avoided direct observation. We found a tendency for males to be sampled more frequently than females, which is consistent with the greater wariness of unhabituated female chimpanzees observed at multiple field sites (Bertolani and Boesch 2008; Johns 1996; Sommer et al. 2004). Given that the area occupied by the Gishwati chimpanzees is only 9 km2, the estimate of 19 individuals implies a density of ca. 2.1 chimpanzees/km2, which is higher than observed at several other nearby montane rain forest sites (Kabira: 0.98/km2, Nyungwe: 0.35/ km2, Barakabuye et al. 2007; Kahuzi: 0.13/km2, Yamagiwa et al. 1992). The small size of the area occupied by the Gishwati chimpanzees makes it improbable that more than one chimpanzee community currently coexists in this forest fragment. Nonetheless, it is possible that the rapid loss of habitat was accompanied by indiscriminate loss of individuals from the multiple communities that once may have been present, leaving surviving representatives or descendants of more than one social group. If that were the case, we would potentially find markedly high Y-chromosome diversity levels in the current Gishwati population due to the presence of male lineages from what once had been multiple communities. However, we found that the level of Y chromosome genetic diversity in the Gishwati community was not larger than that of four other single East African chimpanzee communities, suggesting that the chimpanzees in the Gishwati population, or at least the males, may have all come from a single source community. Although genetic diversity is expected to decrease in a small, isolated population, such effects are not immediate but accrue over time (Lacy 1997; Langergraber 2007a). Therefore, the observation that the genetic diversity observed is similar to that in other chimpanzee communities is consistent with expectations for a population that has been isolated for a relatively short time (Table I, Fig. 2).
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Fig. 2 Relative locations of Gishwati and some other eastern chimpanzee communities. Dots show the approximate locations of the East African chimpanzee study sites mentioned in the text. Black lines depict national borders. The gray areas representing protected areas were derived from the World Database on Protected Areas (WDPA) Annual Release 2009.
The Gishwati chimpanzees’ isolation, however, still threatens their long-term survival. Continued inbreeding and genetic drift could eventually reduce the population’s ability to adapt to environmental change and lead to the buildup of maladaptive traits (Lacy 1997). Currently, the government of Rwanda and the organization Great Ape Trust and Drake University are planning a corridor that will connect the Gishwati chimpanzees to their neighbors in Nyungwe National Park 70 km to the south. Through the use of this corridor, the Gishwati chimpanzees will potentially be able to exchange genetic material with the larger Nyungwe population, increasing their prospects for long-term survival. Once this corridor is implemented, repeated genetic censusing will be valuable for tracking the fates of individuals, including
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understanding how individuals from both populations use the newly available space, detecting instances of migration between populations, and recording reproductive events (Goossens et al. 2003). In this study we obtained a population estimate with reasonable confidence intervals by analyzing ca. 3.5 times as many samples (N068) as individuals found in the population. Although this represents rather intense sampling and laboratory effort, it is worth noting that many samples used in this study were found singly or near few other samples and not in the context of groups of nests (data not shown), suggesting that even more frequent, comprehensive sampling would be needed to draw reliable conclusions on association and ranging patterns of individuals. Information on association, as well as the approximate ages of individuals, would also aid in the assessment of relationships between individuals. Although two individuals that share an allele at each locus may represent a parent–offspring pair, information on approximate ages of individuals greatly aids in the confident identification of mother-father-offspring trios. Thus, a combination of observational and genetic methods would be an effective way to monitor the Gishwati chimpanzees in the future. In summary, this study provides support for the utility of the genetic survey approach. It, along with other studies, shows how DNA from noninvasive sampling can effectively answer demographic questions without habituation of focal animals (Bergl and Vigilant 2007; Bradley et al. 2008; McGrew et al. 2004). This approach is especially useful for genetic sampling of small isolated primate populations, particularly those that have recently been fragmented from larger populations. These populations may likely still retain a high degree of genetic diversity, making them good candidates for conservation strategies such as reconnection to larger populations through the use of forest corridors. Acknowledgments The Great Ape Trust provided funding for this study. We thank the Government of Rwanda for their support of our research in Gishwati Forest Reserve. We also thank Benjamin Beck, Madeleine Nyiratuza, Sylvain Nyandwi, Peter Clay, Thomas Safari, Samuel Uwimana, Patience Mwiseneza, Alex Ndayambaje, Isaac Ngayincyuro, Olivier Ngabonziza, and Eric Munyeshuli for both logistical support and assistance in the field. We especially thank Hjalmar Kühl for assisting with the map. We thank Christophe Boesch and Mimi Arandjelovic for discussion, Roger Mundy for assistance with the accumulation curve estimation, and Carolyn Rowney for assistance with laboratory work. In addition, we thank two anonymous reviewers and Joanna Setchell for comments on previous versions of the manuscript. The Max Planck Society provided funding for laboratory work.
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