Mitochondrial DNA Variability in Grauer’s
Gorillas of Kahuzi-Biega National Park
K. Saltonstall, G. Amato, and J. Powell
Eastern lowland gorillas (Gorilla gorilla graueri) are the least studied of the three
gorilla subspecies, particularly at the molecular level. We sequenced an internal
region of the mitochondrial DNA cytochrome oxidase subunit II (COII) region and
a hypervariable portion of the mitochondrial DNA control region (D-loop) from wild
gorillas in both the montane and lowland habitats of Kahuzi-Biega National Park,
Democratic Republic of Congo. All individuals (n 5 38) were identical at the COII
region; this sequence indicates that diagnostic sites previously suggested for gorilla subspecies may be valid. Low variability was found within the D-loop region
from a subset of the individuals (n 5 15) sequenced for COII. Haplotype frequencies
differed between the two habitats, suggesting a level of population subdivision that
may have demographic consequences. These results also support the distinction
of two distinct clades of gorillas comprised of western populations (G. g. gorilla)
and eastern populations (G. g. graueri and G. g. beringei). Future management of
Kahuzi-Biega National Park should ensure that sufficient habitat remains to prevent
further genetic isolation of gorillas in the montane section of the park.
From the Yale School of Forestry and Environmental
Studies (Saltonstall) and the Department of Ecology
and Evolutionary Biology (Saltonstall and Powell), Yale
University, New Haven, Connecticut, and the Wildlife
Conservation Society, Bronx, New York (Amato). This
project was completed as part of the requirements for
the Masters of Forest Science degree at the Yale School
of Forestry and Environmental Studies. Funding for this
research came from the Wildlife Conservation Society
Research Fellows Program. The acquisition and maintenance of facilities was supported by grants from the
National Science Foundation. We would like to thank
Jefferson Hall and all participants in the Grauer’s Gorilla and Eastern Forest Survey who assisted in hair
sample collection. Tony Goldberg provided advice on
sample extraction procedures and Gisella Caccone
gave technical advice on laboratory procedures. We
thank James Gibbs, Rob DeSalle, and several anonymous reviewers for comments on the manuscript. Address correspondence to K. Saltonstall, Department of
Ecology and Evolutionary Biology, 427 Osborn Memorial Laboratories, P.O. Box 208104, Yale University, New
Haven, CT 06520–8104 or e-mail: kristin.saltonstall@.
yale.edu.
Journal of Heredity 1998;89:129–135
Three subspecies of gorillas are currently
recognized: western lowland gorillas (Gorilla gorilla gorilla), eastern lowland or
Grauer’s gorillas (G. g. graueri), and mountain gorillas (G. g. beringei; Groves 1970).
Of the three subspecies, Grauer’s gorilla is
the least studied. An estimated 8150 individuals (4500–11,800) of this subspecies
inhabit the lowland forest parks and reserves of eastern Democratic Republic of
Congo ( Hart and Hall 1996). Gorillas in all
regions of Africa are threatened by human
encroachment in the forms of hunting for
food, capture of infants for sale, and land
clearing. Eastern Democratic Republic of
Congo has some of the highest human
densities in Central Africa, with a population density of 300 individuals/km2 in
some areas ( Institut National de la Statistique 1984). This human density has recently been augmented by the influx of refugees from Rwanda into eastern Democratic Republic of Congo, creating additional pressures on the available forest
habitat. Such threats have led the IUCN to
declare the graueri subspecies as endangered.
Knowledge of the genetic structure of
populations of endangered species is important for any conservation program targeting individual species (Amato and Gatesy 1994; Avise 1996; Hedrick and Miller
1992). Important genetic issues include
the degree of evolutionary differentiation
and novelty represented by a population,
as well as the relative relatedness of individuals and possible effects of inbreeding
(Amato 1991; Templeton 1986). Little is
known about genetic diversity in G. g.
graueri. Previous studies used limited samples and have indicated an evolutionary
split between eastern and western gorillas
(Garner and Ryder 1992, 1996; Ruvolo et
al. 1994). The degree to which graueri are
evolutionarily distinct from mountain gorillas and estimation of the levels of gene
flow that occur between populations of
the subspecies warrants further study.
Such information is needed to establish
conservation priorities for eastern gorillas.
The issue of intraspecific variability in
hominoids has been well studied. For gorillas, molecular and morphological data
currently support two distinct clades—
one composed of western lowland populations and the other including the two
eastern subspecies (Garner and Ryder
1992; Ruvolo et al. 1994). Much of this
work has been done, however, using samples from zoo animals whose origins are
unknown. Of the three subspecies,
Grauer’s gorilla has been the least represented in such studies, presumably due to
129
their scarcity in zoos and the lack of field
research focused on the subspecies. Recently developed techniques using hair
samples for DNA extraction ( Higuchi et al.
1988; Morin et al. 1993; Woodruff 1993)
and the polymerase chain reaction (PCR;
Innis et al. 1988) now allow wild individuals to be studied using noninvasive techniques for sample collection.
Although consensus has been reached
that all gorilla populations west of the African Lakes are distinct from the mountain
gorillas inhabiting the Virunga Volcano region (Groves and Stott 1979), the systematic designation of individual populations
under the name of ‘‘eastern gorilla’’ has
been subject to much debate. One such
population inhabits the forests surrounding Mt. Kahuzi in Kahuzi-Biega National
Park. Groves (1967, 1970) assigned them
to the beringei subspecies based on postcranial features but considered them to be
transitional between the true graueri of
the nearby Utu lowland region and the beringei populations of the Virunga region. In
revisiting this designation, Groves and
Stott (1979) concluded that the Kahuzi gorillas belong to the subspecies graueri but
have some beringei features, suggesting
that at some time in the past they were in
reproductive contact with the Virunga
population but since then have been more
influenced by the true graueri of the Utu
region. This classification holds today and
indicates that clinal variation may exist
between gorillas living in different habitat
types. Morphological differences between
the gorillas in the montane section of Kahuzi-Biega National Park and those in the
lowlands are well documented, but these
populations have never been examined genetically (Groves 1970; Groves and Stott
1979). This study compares patterns of
variation in mitochondrial DNA between
the two populations.
We amplified DNA from hair samples obtained from wild gorillas in Kahuzi-Biega
National Park. Due to the degraded state
of many of the samples, we amplified only
short regions (250–300 base pairs) of DNA.
We conducted an analysis of a 252 base
pair internal region of the mitochondrial
protein-coding gene for cytochrome oxidase subunit II (COII) and a 277 base pair
hypervariable portion of the mitochondrial DNA displacement loop ( D-loop) control region to examine patterns of genetic
diversity within the graueri subspecies
and for comparison with other gorilla subspecies. First, we used the mtDNA data to
evaluate the hypothesis that within the
different habitats of Kahuzi-Biega National
130 The Journal of Heredity 1998:89(2)
Figure 1. Map of Kahuzi-Biega National Park and the surrounding area. Zone I refers to the lowlands and zone
II represents the montane sector of the park.
Park, gorillas may have different histories
of reproductive contact with other populations. Second, we compared diversity
within graueri to other subspecies of gorilla and examined it in relation to previously suggested diagnostic sites which indicate genetic differentiation from other
gorilla subspecies (Garner and Ryder
1996; Ruvolo et al. 1994). Finally, we discuss the implications of these results for
Grauer’s gorilla conservation and management.
Materials and Methods
Study Area
Kahuzi-Biega National Park is located in
the Kivu region of eastern Democratic Republic of Congo (278339 288519 west and
18529 28299 south; Figure 1). The park is divided into a 600 km2 montane sector and
a 5400 km2 lowland sector which are connected by a narrow corridor. Although it
is forested, the corridor is surrounded by
a dense human population and the degree
to which it is used by gorillas is unknown.
In the past solitary gorilla nests have been
found in the corridor region (Refisch
1991), however, today forest cover has
been destroyed and much of the area is
used for agriculture ( Inogwabini et al., in
preparation). We divided the park into two
sampling zones: zone I in the lowland extension of the park and zone II in the montane sector of the park ( Figure 1).
Sample Preparation
Hair samples were collected from vacated
day or night nests of wild individuals and
stored in paper envelopes at ambient temperatures. Samples from a total of 16 family groups in zone I and 3 family groups
( Nindja, Maheshe, and Mushamuka) in
zone II were sequenced ( Table 1). Samples
in zone I were collected during a survey
of large mammals in 1994 in which line
transects were walked and gorilla nest
sites identified and mapped. The nature of
the survey technique and the means of
data collection allowed gorilla groups to
be identified based on group size. Samples
collected from nest sites thought to have
been created by the same family group
were not included in this analysis in order
to avoid duplicate sampling of individuals.
In some cases, multiple individuals from a
family group were sequenced to increase
the total sample size ( Table 1). Where
multiple individuals within a group were
found to have the same haplotype for the
loci examined here, only single represen-
quencing reactions were performed using
the Dye Terminator Cycle Sequencing kit
(Perkin Elmer) and an automated sequencer (ABI 373). Samples were sequenced in both directions and individuals were sequenced twice to detect Taq
DNA polymerase errors. Where differences
were observed, individuals were resequenced from new PCR products to resolve any discrepancies.
Table 1. Family group, identification, and geographic origin of Gorilla gorilla graueri individuals in
Kahuzi-Biega National Park
Family group
Individuals
Zone
Location
A
B
C
D
E
F
G
H
I
J
K
L
M
N
O
P
Mushamuka
Nindja
Maheshe
G11, G14
G115
G136
G142, G143
G23
G27
G212
G216
G219
G223
G232
G31
G33
G34
G39
G413, G417, G420
G51, G52, G53, G54, G55
G57, G59, G510, G513, G514, G515, G516, G517, G520
G524, G525, G528, G530, G531
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
II
II
II
Busakala
Busakala
Busakala
Busakala
Mankese
Mankese
Mankese
Mankese
Mankese
Mankese
Mankese
Mankese
Mankese
Mankese
Mankese
Nzovu
Tshivanga
Tshivanga
Tshivanga
tatives of each group were used for comparisons.
Extractions were performed using 250
ml 5% Chelex solution, with 1.5 ml 20
mg/ml proteinase K and 7 ml 1 M DTT
(Goldberg T, personal communication;
Walsh et al. 1991). One to five hairs were
used per individual, depending on the
number of hairs collected. Following overnight digestion at 568C, samples were vortexed, placed in a boiling water bath for 8
min, and then centrifuged for 3 min so that
the resulting supernatant could be removed. The Chelex resin was then resuspended in 200 ml of water, vortexed, and
centrifuged again to elute any remaining
DNA. The resulting supernatant was removed and added to the previous solution. This final DNA template was concentrated using Microcon 30 microconcentrators to a volume of 20 to 50 ml.
PCR Conditions
The polymerase chain reaction (PCR) was
used to create double-stranded DNA using
Results
A total of 39 individuals were sequenced
for the COII gene, all of which were identical. Of these 39, 20 (representing 16 family groups) came from the zone I lowlands
and 19 (representing three family groups)
from the montane zone II. This sequence
is also identical to the one obtained by Ruvolo et al. (1994) for the graueri subspecies, based on sequencing of one zoo individual of unknown origin. The sequence
differs from the other eastern gorilla subspecies (G. g. beringei) at two positions
(Ruvolo et al. 1994; Table 2a).
D-loop sequences were obtained from
15 individuals. These samples represent a
subset of those analyzed with the COII
gene, with 5 from zone I and 10 from zone
II ( Table 3). Although attempts were made
to obtain sequences from all individuals at
this region, the degraded nature of the
samples prevented us from successfully
amplifying all of them. Six mtDNA haplotypes were found, differing by 0.4 to 1.8%
( Figure 2 and Table 4). Of these, haplotypes designated EL1, 2, 3, and 6 were previously identified in another study, which
also found two other haplotypes, here
designated as EL5 and EL8, respectively
( Table 4; Garner and Ryder 1996). This
analysis also includes a variable internal
region of approximately 30 base pairs cen-
reaction conditions described by Innis et
al. (1988). Primer sequences for the COII
fragment were C187G: 59 TCAGACGCCCAAGAAATAGAGA 39, and D402G: 59
TCGGTTGTCGACGTCAAGGAGT 39. These
primers were modified from those used by
Ruvolo et al. (1994) for specificity with G.
g. graueri based on published sequence
(Ruvolo et al. 1994). Primer sequences for
the D-loop region are described in Garner
and Ryder (1996). The reaction cycle consisted of denaturation at 948C for 30 to 60
s, annealing at 508C–558C for 30 to 60 s,
and DNA extension at 728C–748C for 1 to 5
min. Samples were amplified for 40 cycles.
In most cases, reamplification using 1 ml
of PCR product was necessary to produce
sufficient quantities of DNA for sequencing.
DNA Sequencing
PCR products were purified for sequence
analysis with the GeneClean III kit
( Bio101) using the directions supplied by
the manufacturer. Single-stranded se-
Table 2. Diagnostic positions which distinguish the three subspecies of gorilla (A) within COII, (B) within D-loop
(A)
WL
MT
EL
a
22
39
72
105
108
117
181
192
195
204
A
G
G
C
A
A
T
C
C
T
C
C
C
C
T
C
T
T
A
A
G
C
T
T
T
C
C
C
T
T
( B)
WL
MT
EL
b
1
11
17
27
53
105
106
117
122
123
124
135
136
141
143
144
145
151
153
154
161
199
220
226
241
A
G
G
A
G
G
C
C
T
C
T
T
G
G
T
A
C
C
A
C
C
C
T
T
A
T
T
C
—
—
A
—
—
T
C
C
C
A
A
C
—
—
C
A
A
C
T
T
C
T
C
T
T
C
T
A
A
G
C
C
G
G
A
A
A
G
A
C
C
C
T
C
T
C
C
Site numbers refer to positions within the gene.
Western lowland (WL) gorilla (Gg04; Ruvolo et al. 1994). Substitutions are shown in mountain gorilla (MT; Gg06; Ruvolo et al. 1994) and eastern lowland gorilla ( EL; Gg05;
Ruvolo et al. 1994; this study) sequences.
b
Reference sequence is a western lowland gorilla (WL191; Garner and Ryder 1996). Mountain and eastern lowland gorilla substitutions are based on sequences described in
Garner and Ryder (1996) and this study.
a
Saltonstall et al • Mitochondrial Variation in Grauer’s Gorillas 131
Table 3. D-loop haplotypes identified for
Grauer’s gorillas
D-loop
haplotype
EL1
EL2b
EL3c
EL4
EL6d
a
EL7
Individual(s)
G223
G31
G115
G33
G34, G51, G54, G59, G513, G514,
G515, G516, G517, G530
G531
Corresponds to
Corresponds to
c
Corresponds to
d
Corresponds to
a
b
EL058
EL094
EL055
EL099
(Garner
(Garner
(Garner
(Garner
and
and
and
and
Ryder
Ryder
Ryder
Ryder
1996).
1996).
1996).
1996).
tering on a string of C’s not included in the
phylogenetic analysis by Garner and Ryder (1996). Because gorillas are the species of interest here and intraspecific
alignment of this region is possible, we included it in our analysis.
The six D-loop haplotypes identified
here were not distributed randomly
throughout the two study zones. Haplotype EL6 predominated in zone II and was
found in all three family groups that were
sampled. One individual in zone I also carried this sequence ( Table 3). One other sequence, EL7, was found in one individual
of zone II.
The remaining four haplotypes described here were each found in one individual from zone I ( Table 3). Three of
these ( EL1, 2, and 3) were previously identified by Garner and Ryder (1996), who
also found one other sequence ( EL5) in
this habitat region that was not identified
in this study.
Discussion
Genetic Differences Within Gorillas in
Kahuzi-Biega National Park
Previous morphological studies on the gorillas in Kahuzi-Biega National Park have
distinguished the montane from the lowland populations on the basis of both cranial and postcranial indices (Casimir 1975;
Goodall and Groves 1977; Groves 1970;
Groves and Stott 1979). Inclusion of the
montane population under both the
graueri and beringei designations has been
called for, however, it has never been suggested that gorillas in the montane sector
be given a subspecies rank despite their
distinctive
morphological characters
(Groves and Stott 1979). This study affirms this conclusion by showing that mitochondrial genetic data clearly links the
two populations.
The protein-coding COII region is invari-
132 The Journal of Heredity 1998:89(2)
Figure 2. Alignment showing variation in the eastern lowland gorilla mitochondrial D-loop gene within a 277
base pair internal fragment. Sequences EL5 and EL8 are from Garner and Ryder (1996).
ant between the study zones indicating
relatively recent common ancestry for the
two populations. It also shows no difference with previously published sequences
for this gene in a graueri individual of unknown origin (Ruvolo et al. 1994). In that
study, graueri were found to differ from
beringei at only two sites in the region examined here ( Table 2a). Using a moderately conserved gene region such as COII results in fewer variable sites for character
analysis (Ruvolo et al. 1993). However, it
Table 4. Percent nucleotide sequence differences between a 277 base pair region of D-loop sequences determined from mountain and eastern lowland
gorillas
MT045
MT074
MT374
MT504
EL1
EL2
EL3
EL4
EL5
EL6
EL7
EL8
MT045
MT074
MT374
MT504
EL1
EL2
EL3
EL4
EL5a
EL6
EL7
EL8b
—
1.1
—
1.4
0.4
—
1.1
0.7
1.1
—
6.1
7.2
7.6
6.5
—
6.9
7.9
8.3
7.2
0.7
—
6.1
7.2
7.6
6.5
0.7
0.7
—
6.1
7.2
7.6
6.1
0.7
0.7
0.7
—
6.5
7.6
7.9
6.9
1.1
1.1
0.4
1.1
—
6.1
7.2
7.6
6.5
1.4
1.4
1.4
0.7
1.8
—
6.5
7.6
7.9
6.9
0.4
0.4
0.4
0.7
0.7
1.1
—
7.2
7.9
8.7
7.6
1.1
1.1
1.1
1.1
1.4
1.8
0.7
—
All mountain gorilla samples are of wild origin (Garner and Ryder 1996). Samples EL1–EL5 are wild individuals living in the zone I lowlands of Kahuzi-Biega National Park.
EL6 and EL7 are wild individuals living in the montane zone II. EL8 is a captive individual of unknown origin (Garner and Ryder 1996; this study).
a
Corresponds to EL086 (Garner and Ryder 1996).
b
Corresponds to EL057 (Garner and Ryder 1996).
is likely that the two phylogenetically diagnostic characters supported by this expanded dataset are less likely to be affected by problems with homoplasy than are
characters in other regions, such as the
hypervariable D-loop region (Amato 1994;
Amato et al. 1995; Ruvolo 1994). For these
reasons, inclusion of the population in the
montane zone II in the graueri designation
rather than beringei seems warranted.
In contrast to the COII fragment, the hypervariable portion of the D-loop control
region analyzed here showed several base
changes throughout the samples ( Figure
2). This region has previously been observed to evolve on the order of 10 times
faster than COII (Garner and Ryder 1996;
Ruvolo et al. 1993). Several haplotypes
were found, but their distribution and frequency varied by zone. Zone II was dominated by sequence EL6 (all groups, 9 of 10
individuals), previously identified by Garner and Ryder (1996). While only three
family groups were sampled here, it is
probable that a greater number of matrilineal lines are actually represented here
due to female transfer between groups.
Recent census information indicates that
the gorillas in this region of the park are
now confined to a 200 km2 central region
due to human encroachment ( Inogwabini
et al., in preparation; Vedder et al., unpublished data). Within this small area, at
least 25 groups are found ( Vedder et al.,
unpublished data), making it likely that
contact between groups occurs regularly.
Thus EL6 may be widespread throughout
zone II.
Evidence for contact between gorillas in
both zones also exists. Sequence EL6 also
appeared in a lone male sample (G34) collected in zone I. Further, Garner and Ryder
(1996) identified sequence EL2 in an individual from the Mushamuka group found
in zone II. In our study, we found this sequence in a lone male from zone I (G31).
This suggests that in the recent past, if not
still today, gorillas have traversed the corridor connecting the montane and lowland
habitats bringing the two populations into
reproductive contact. Given the population size of the montane area (n 5 243;
Vedder et al., unpublished data), had gene
flow ceased between the two populations,
greater subdivision between the two
zones might have occurred due to genetic
drift.
Diversity Within and Differences
Between Gorilla Subspecies
An issue of importance for both conservation and evolutionary biologists regarding variability within a species is that of
variation within populations and between
populations. Without knowledge of sample
origin, it is impossible to make such distinctions. Ruvolo et al. (1994) have distinguished four haplotypes within western
lowland gorillas based on COII sequences
from zoo individuals of unknown origin.
Variability in both mountain and eastern
lowland gorillas at the COII gene appears
to be much lower than in western gorilla
populations (Ruvolo et al. 1994). Today
graueri are found in 11 distinct populations which have little or no contact with
each other due to human presence ( Hall
et al., in press a) while mountain gorillas
are found in only two populations which
also are geographically isolated. Within
graueri, this study demonstrates that little
to no variability exists at COII among the
gorillas of Kahuzi-Biega National Park, but
no statement can be made about levels of
variation within the subspecies as a
whole. Among mountain gorillas the same
holds true. Within western lowland gorillas, recent data have shown variability
within regional populations to be much
less than in the subspecies as a whole
(Garner and Ryder 1996). Until more geographically isolated populations have
been sampled, the level of differentiation
at the molecular level within eastern gorilla populations remains unclear. For conservation purposes, correlation of genetic
variation with geographic location is essential (Amato and Gatesy 1994; Avise
1989).
These results also support previous
designations of phylogenetic clades in the
gorilla species (Garner and Ryder 1992;
Ruvolo et al. 1994). Based on an earlier
study using COII sequences from six individuals (four western lowland, one eastern
lowland, and one mountain gorilla; Ruvolo
et al. 1994), diagnostic sites for the three
subspecies of gorilla may be identified
( Table 2a). Eastern gorillas (graueri and
beringei) can be distinguished from western gorillas (gorilla) at eight positions.
Two additional sites are unique to graueri.
This study greatly expands the dataset for
eastern lowland gorillas at this site by
confirming marker positions for the two
populations examined here. However, additional study on other populations of
Grauer’s gorilla that are geographically
isolated is warranted to validate the sites
for the subspecies as a whole.
Garner and Ryder (1996) identify a number of positions within the D-loop fragment that are consistent within the subspecies based on their samples. Our results also concur with their findings. Eastern gorillas can be distinguished from
western gorillas at 18 positions within this
region. The graueri subspecies can be distinguished from beringei at another seven
positions ( Table 2b). Differences between
individual eastern gorillas range up to
8.7% in this region ( Table 4), making the
Saltonstall et al • Mitochondrial Variation in Grauer’s Gorillas 133
D-loop a more useful mitochondrial region
for estimation of genetic variability between populations of a subspecies. While
these diagnostic positions are based on
limited samples from two known graueri
populations as well as several individuals
of unknown origin, they provide information on regional if not subspecific differentiation for Grauer’s gorillas. These diagnostic markers could be extremely useful in identifying the origins of animals
captured for illegal trade (Garner and Ryder 1996).
Management Implications
Our results clearly demonstrate that despite their morphological differences, the
gorillas inhabiting the montane and lowland regions of Kahuzi-Biega National Park
are closely related. In spite of the intense
human pressure in the regions surrounding them, gorillas in the two habitat
regions are possibly in reproductive contact even today. However, the levels of
contact may be low and may not be significant from a demographic perspective.
Low levels of gene flow and dispersal
rates, as well as geographical structuring
in mtDNA, can imply demographic independence among populations (Avise
1995). This is particularly true in species
such as gorillas where females, although
they may transfer between family groups
( Yamagiwa 1983), do not travel great distances to mate ( Harcourt 1978). The only
D-loop sequences common to both zones
were found in lone males from zone I (this
study; Garner and Ryder 1996). Within Kahuzi-Biega, gorilla populations in the two
study zones are separated by nonforested
land that is inhabited and used by humans. Such breaks in forest cover probably hinder movement of gorillas between
populations. Dispersal between the gorilla
populations of zones I and II appears to be
occurring at an evolutionarily significant
level of gene flow. However, at the demographic level it may not be enough to sustain the small montane population in the
long term, due to low recruitment rates of
females from the nearby lowlands (Avise
1995; Taylor and Dizon 1996).
Despite this, every effort should be
made to restore and maintain the corridor
connecting the two sections of the park so
that gorillas, and other forest dwelling
species, may continue to travel between
the two habitats. Since the montane population is one of the few gorilla populations known to have remained relatively
stable in size (Murnyak 1981; Vedder et al.,
unpublished data; Yamagiwa et al. 1993)
134 The Journal of Heredity 1998:89(2)
and the lowland population makes up the
core of the largest population of Grauer’s
gorillas today ( Hall et al., in press b), it is
critical that they remain protected.
Noninvasive Sampling
Finally, we would like to comment on the
difficulties of working with hair samples,
especially samples such as those analyzed
in this study that have been exposed to
the elements and are in a somewhat degraded state. Because of the difficulties in
obtaining adequate amounts of DNA, the
sample size analyzed here is small. DNA
extractions were attempted from 104 hair
samples, but sequencing was possible in
only 39 of these samples. Length of time
before collection of hair samples appears
to be important in the success of DNA amplification, as samples from the montane
area which were collected from nests created by gorillas the previous night had
much higher rates of successful amplification than nests from the lowlands where
length of exposure was not known. However, several samples for which the age of
the nest from which they were collected
was estimated to be 4 to 6 weeks were successfully amplified and sequenced, indicating the usefulness of this noninvasive
method of obtaining DNA samples from
wild individuals.
This dataset is the largest to analyze relationships between graueri populations.
Since all of the individuals tested here are
also of known origin, we have been able
to draw some conclusions about the extent of variability within gorillas of KahuziBiega National Park. With threatened or
protected species, such data are essential,
as are noninvasive methods of obtaining
samples. Further studies are necessary to
assess the variability of the subspecies as
a whole as well as to confirm the diagnostic marker positions that we identify. Molecular studies of mitochondrial as well as
nuclear DNA (e.g., microsatellites) can
provide relevant information to a range of
disciplines, including conservation and
management, evolutionary biology, and
taxonomy.
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Received December 4, 1996
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Corresponding Editor: Stephen J. O’Brien
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