Mitochondrial DNA Variation in Western Lowland Gorillas (Gorilla gorilla gorilla)




C. A. Corky Gaines

Introduction

The western lowland gorilla is one of three recognized subspecies of gorillas. Inhabiting several countries in western Africa, the current population of western lowland gorillas in the wild is estimated at between 30,000 and 45,000. They are currently listed as endangered by the IUCN - The World Conservation Union. The stability of the population is in jeopardy, due to habitat loss and poaching, with a population reduction of 50% predicted within three generations (13).

Captive western lowland gorillas have gone through a severe founder effect. 112 founders have contributed genetically to the current North American population of approximately 400 individuals. As expected, not all of these founders have contributed equally. A Gene Drop Analysis produced a Founder Genome Equivalent of approximately 45 (24).

Founder effects and population bottlenecks can have very strong genetic consequences. These include a reduction in effective population size, a loss of heterozygosity, fixation of alleles and expression of deleterious recessive alleles. Ultimately, all of these consequences can lead to a strong reduction in fitness and survival (10). In addition to the initial loss of genetic diversity, genetic drift is enhanced in smaller populations, leading to increased fixation of alleles.

Captive western lowland gorillas provide a unique opportunity to study the genetic diversity of the population prior to and following the bottleneck. Several studies have attempted to measure the consequences that a founder effect or a bottleneck effect, can have on the genetic variation of a population (1, 3, 10, 18, 23, 26). While these studies are able to measure the current genetic diversity, most are unable to measure genetic diversity prior to the onset of the bottleneck, or can only estimate genetic diversity from a few museum samples (10).

This study set out to develop a protocol that would allow for non-invasive sampling of DNA and provide DNA sequences with variation at the population level. Fecal matter can contain intestinal epithelial cells and has proven useful in obtaining DNA from several species (5, 9, 14, 19).

The mtDNA of several species have been used to study the genetic variation of species at the population and species level (3, 8, 10, 22). The mtDNA of gorillas is a circular DNA, approximately 16 kb long which contains a D-loop control region approximately 800 bp (2). The mtDNA is ideal for population level studies for several reasons. The mtDNA of primates has been measured to evolve 5-10 times faster than nuclear DNA and even faster in the D-loop, which is a non-coding region (4) and apparently free from the effects of natural selection (7). This charactersitic can lead to intraspecies variation. Beyond closely related species, mtDNA variation is likely to be obscured by multiple substitutions at the same site (17). In addition, mtDNA is maternally inherited and undergoes recombination rarely, if at all (11). Finally, hundreds of copies of mtDNA, can be found in each cell (16). Care must be taken when using mtDNA as a reference for diversity of the entire genome of a species. The mtDNA is a small part of the entire genome and trends in the mtDNA may not reflect trends in other areas of the genome. However, differences between the trends of the mtDNA and nuclear DNA may be less in Hominoids due to female dispersal (21).

This study provides a protocol for retrieval of mtDNA from the fecal samples of western lowland gorillas and shows that variation can be detected at the population level.

Figures


Figure 1- Amplification of a 335 base pair segment of the mitochondrial DNA D-loop. A) Lanes 2-5 and 7-10 included isolated DNA diluted to 50 ng/uL and 25 ng/無 respectively, for BZ1 (lanes 3 and 8), BZ2 (lanes 2 and 7), BZ3 (lanes 4 and 9), and BZ4 (lanes 5 and 10). Lanes 1 and 6 represent negative controls. Lane 11 is a 100-bp ladder. B) Lanes 2-3 and 4-5 included isolated DNA diluted to 10 ng/無 and 5 ng/無 respectively, for BZ3 (lanes 2 and 4), and BZ4 (lanes 3 and 5). Lane 1 is a negative control and lane 6 is a 100-bp ladder. DNA was amplified according to Materials and Methods and visualized on ethidium bromide stained agarose gels.


Figure 2-Partial sequence for mitochondrial DNA D-loop region for two western lowland gorillas, BZ1 and BZ2. DNA was sequenced according to Materials and Methods. Arrows indicate locations of base-pair differences and numbers refer to base-pair location in figure 3.


Figure 3-Aligned sequences of BZ1, BZ2, and BZ3 to a previously published sequence for western lowland gorilla (Gorilla gorilla gorilla), from Genbank, using Macvector.


RESULTS

mtDNA D-loop sequences from two of the three Bronx Zoo individuals (BZ1, BZ2) are shown in figure 2, and are aligned to a previously published sequence of a western lowland gorilla (figure 3). The gorilla mtDNA D-loop region contains at least one deletion when compared to the human mtDNA control region. The deletion(s) appears to correspond to positions 122 through 225, of the human sequence (8). Located in this portion of the gorilla sequence are two strings of C’s which flank two T’s. These strings of C’s are highly variable in gorillas and appear to be highly susceptible to Taq polymerase amplification errors. In a study by Garner and Ryder (1996), several sequences from the same individual would reveal differing numbers of C’s in this region. This region and the region corresponding to positions 122-225 of the human sequence were excluded from analysis of the Bronx Zoo individuals.

The three Bronx Zoo individuals were found to contain two different nucleotypes. The sequence identified as BZ1 in figure 3, is identical to the previously published sequence (Western Lowland Gorilla) for positions 1 through 274. Several positions beyond 274 are undetermined in both sequences. The other two sequences, BZ2 and BZ3 are identical to each other not including four positions in BZ2 and a single position in BZ3, that were not determined. BZ2 and BZ3 differed from Western Lowland Gorilla and BZ1 by eight transitions and one transversion (3.2% of the total sequence). One of the transitions, corresponding to position 145 is not conclusive. The transition from T to C may actually be a deletion or insertion of the T, if the C’s in BZ2 and BZ3 are considered part of the second string of C’s previously excluded from the analysis.

DISCUSSION

Development of a Suitable Protocol

This study showed that amplifiable DNA can be retrieved from fecal samples of western lowland gorillas, as has been shown for other species. Several studies have shown variable success rates in amplifying DNA from fecal samples (6, 14, 20). These studies either did not measure the amount of DNA used during PCR reactions or did not indicate if measured amounts were used. In this study, undiluted DNA, containing between 100 and 250 ng/無, were added to the PCR mixtures. These first mixtures produced no amplified PCR product (data not shown). A second and third set of PCR reactions included isolated DNA diluted to 50 and 25 ng/無, respectively. None of the samples produced amplified PCR product at 50 ng/µL. However, two of the samples, BZ1 and BZ2 produced amplified DNA product at 25 ng/無 (figure 1a). Finally, dilutions of 10 and 5 ng/無 were included in PCR mixtures for BZ3 and BZ4, and produced amplified DNA at both dilutions (figure 1b). In light of the fact that very little DNA is needed for successful amplification, it is recommended that isolated DNA be diluted to small quantities in order to minimize the effects of PCR inhibitors.

Implications for Future Study

Most wildlife parks today practice a "hands-off" approach to care for captive animals, in an attempt to minimize human-animal interaction, not wanting to alter their natural behaviors. The same can be said for studies of animals in the wild. In the past, genetic studies required behavioral conditioning or sedation in order to obtain blood samples (8). The advent of obtaining DNA from waste products, proved to be a non-invasive technique with varying success. If this simple protocol for fecal DNA extraction proves to have a high success rate, it can lead to complete studies of genetic variation in western lowland gorillas and other species.


Acknowledgments

I would like to extend my sincerest thanks to Sabrina Volpi and Rocco Coli, who taught me the techniques necessary for this project. I would also like to thank Dr. Berish Rubin, for the opportunity to complete this project and for the use of his laboratory. Finally, thanks to the Wildlife Conservation Society for providing the samples employed in this project.


This document was last modified 01/31/2006.
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