Such variation is important in preserving allelic variations that may be valuable in response to environmental perturbations. Previous studies of allozyme loci have indicated very few polymorphic loci and reported a high degree of homozygosity for Central American howling monkeys (Malmgren and Bush, 1978; Pope 1995). High levels of homozygosity in remnant or island populations such as the Ometepe monkeys may render the population more susceptible to demographic and environmental stressors (Pope 1995). The present study begins an evaluation of heterozygosity in the Ometepe howling monkey population in an attempt to evaluate their conservation status. It utilizes microsatellite loci (using human primers and PCR analysis) which have been identified as polymorphic in a combined sample of Alouatta palliata from Mexico, Costa Rica, Panama (Ellsworth and Hoelzer 1998) and Saimiri (Witte and Rogers, 1999).
Project Goals Several project goals have been identified: Evaluating the physical health status and disease exposure of the howling monkeys on the island of Ometepe;
Tagging and marking the animals for long-term follow-up and behavioral studies;
Obtaining blood samples for analysis of DNA in order to a. Evaluate intragroup heterogeneity b. Evaluate intergroup heterogeneity (long term goal).
Materials And Methods
The study was undertaken at the Ometepe Biological Field Station (110 24' N, 85030' W) on Isla de Ometepe in Lake Nicaragua. The area around the field station is comprised of dry deciduous tropical forest heavily fragmented by land use and deforestation. A group of monkeys (Alouatta palliata) whose behavior had been observed during a previous field season (by L.A.W) was selected for this project. The group was composed of twenty-one animals (5 adult males, 8 adult females, 8 immature) with an infant-juvenile/adult female ratio equal to .63. Their home range was slightly greater than fifteen hectares and composed of patches of secondary forest interspersed with secondary successional growth at various stages of succession. The majority of the adults from this monkey group as well as one subadult (N=11, all five adult males, five adult females) were captured using the Pneudart) system with Telazol) as the capture drug (per Glander et al., 1991) over a six day period in late July, 1998 (see Peter et al. 1999, elsewhere in this symposium for details). Blood was drawn from the femoral vein after which each animal was marked with color-coded collars (E) or ankle bands (G) and ear tags before release (Table 1). DNA was extracted from the blood, isolated and frozen prior to analysis. PCR techniques were utilized to examine 11 microsatellite loci, which had been previously identified for Alouatta palliata and Saimiri (Ellsworth and Hoelzer 1998, Witte and Rogers 1999). We successfully amplified 10 of these loci (see Figure 1) using the following PCR conditions : DNA 40-80ng, dNTP 100 mM, MgCl2 1.5 mM, KCl 50mM, Tris 10mM, Triton 100 0.1%, Primer 0.2 mM, Taq Polymerase 0.5 mM. One primer was radiolabeled with 8-32p using T4 polynucleotide kinase.
Thermal cycle routines were as follows:
1 cycle 5 min @ 95: C
35 cycles 1 min @ 95: C
1 min @ 55: C
1 min @ 72: C
1 cycle 5 min @ 72: C
Results
Our results indicated greater homozygosity for the microsatellite loci than reported for Alouatta palliata from elsewhere (Ellsworth and Hoelzer 1998). As indicated in Table 2, two of these loci had previously been identified as monomorphic in the mantled howling monkey and our results are consistent with that. However, Ellsworth and Hoelzer (1998) identified the remaining eight as polymorphic with allele number as high as six per locus for several. We found only five of these to be polymorphic (see Table 2) with much lower allelic variation. Part of the difference may be in the composition of our samples. Part of the Ellsworth and Hoelzer data is based on combined samples from Costa Rica, Mexico, and Panama (Ellsworth, personal communication) whereas our sample is from a discreet geographic group. In addition, our small sample size may not be representative of potential allelic variation in howling monkey populations elsewhere on Isla de Ometepe or mainland Nicaragua. Although the monkeys within our group are monomorphic and homozygous for 5 of the microsatellite loci, they are polymorphic for the rest. Forty-four % of the alleles were heterozygous from the polymorphic microsatellite loci.
Discussion
A high degree of homozygosity has been reported in other Central American howling monkey populations (Pope 1995). This has been attributed to population crashes due to natural disasters and disease (Crockett 1998). Human interference has also been seen as important in effecting population characteristics of howling monkeys. We anticipated that the isolation of the howling monkeys on Ometepe may increase homozygosity due to genetic drift and island bottleneck phenomena. However, we also anticipated more intragroup variability relative to other howling monkey species since both sexes disperse from their natal group (Glander 1992) in the mantled howling monkey. Nevertheless, the fragmentation of their environment may make it difficult for these monkeys to migrate any substantial distance form their natal group on Ometepe. We did find less allelic variation than reported in howling monkeys elsewhere in Central America (Table 2). But as discussed above, that may be indicative of differences in geographic sampling. It remains unclear whether our small sample is representative of all howling monkeys on the island.
Conservation genetics has generally focused on maintaining overall levels of genetic variation (Hoffman and Parsons, 1991). Genetic variation is important in preserving allelic variants, which may facilitate individual response to severe or prolonged environmental stressors. Overall, genetic variability is dependent on the size and number of animals, intergroup variability of fragmented populations, the degree of fragmentation, and whether fragmentation limits intergroup migration. It is unclear how much the fragmentation which has occurred on Ometepe limits gene flow or overall variability. And as Pope has pointed out, intergroup variability may still exist therefore genetic differences between groups may still be higher than continuous populations.
Conclusions
Further research is necessary to determine the degree of genetic diversity of the howling monkey population on Ometepe. In addition, a plan of conservation and land control needs to be formulated to curb the increasing erosion of the natural resources that are necessary for the monkey's survival. The Ometepe Foundation is currently working with the Nicaraguan government in trying to formulate and implement such a plan. Acknowledgements We would like to thank Rex Sohn and Greg Peter for their collaboration in the field portion of this research (Figure. 5). Their skill, field expertise, and equanimity were critical and much appreciated. And our many thanks to the students and teaching assistants of the advanced primate behavior and biology class (Figure. 5) who served as field assistants, shared their ideas, and in some cases, their photographs. A special thanks you to Toran Hanson who so generously shared his howling monkey photographs. We thank and acknowledge the government of Nicaragua who gave us permission to do this research. And our bounteous gratitude to the Ometepe Biological Field Station, its staff, and the wonderful Molina family who is always hospitable and has offered their support in countless ways. We gratefully acknowledge support from the University of Pittsburgh and Parke-Davis Research. |
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