Molecular Ecology (2004) 13, 15511565

doi: 10.1111/j.1365-294X.2004.02140.x

Mitochondrial DNA phylogeography of western lowland

gorillas (Gorilla gorilla gorilla)

Blackwell Science, Ltd

S T E P H E N L . C L I F F O R D ,* N I C O L A M . A N T H O N Y , M I R E I L L E B A W E - J O H N S O N ,* K A T E
A . A B E R N E T H Y ,* C A R O L I N E E . G . T U T I N ,* L E E J . T . W H I T E , M A G D E L E N A B E R M E J O ,
M I C H E L L E L . G O L D S M I T H ,** K E L L E Y M C F A R L A N D , K A T H R Y N J . J E F F E R Y , M I C H A E L
W . B R U F O R D and E . J E A N W I C K I N G S *
*Centre International de Recherches Mdicales, Franceville (CIRMF), BP 769, Franceville, Gabon, School of Biosciences, Cardiff
University, Cardiff CF10 3TL, UK, Department of Molecular and Biological Sciences, Stirling, FK9 4LA, UK, WCS, Bronx Zoo,
185th Street and Southern Boulevard, Bronx, NY10460-1099, USA, ECOFAC, B.P. 15115, Libreville, Gabon, **Department of
Environmental and Population Health, Tufts University School of Veterinary Medicine, 200 Westboro Road, North Grafton, MA
01536, USA, The Graduate Center, The City University of New York, 365 Fifth Avenue, New York, NY 10016-4309, USA

Abstract
The geographical distribution of genetic variation within western lowland gorillas (Gorilla
gorilla gorilla) was examined to clarify the population genetic structure and recent evolutionary history of this group. DNA was amplified from shed hair collected from sites across
the range of the three traditionally recognized gorilla subspecies: western lowland (G. g.
gorilla), eastern lowland (G. g. graueri) and mountain (G. g. beringei) gorillas. Nucleotide
sequence variation was examined in the first hypervariable domain of the mitochondrial
control region and was much higher in western lowland gorillas than in either of the other
two subspecies. In addition to recapitulating the major evolutionary split between eastern
and western lowland gorillas, phylogenetic analysis indicates a phylogeographical division
within western lowland gorillas, one haplogroup comprising gorilla populations from eastern
Nigeria through to southeast Cameroon and a second comprising all other western lowland
gorillas. Within this second haplogroup, haplotypes appear to be partitioned geographically
into three subgroups: (i) Equatorial Guinea, (ii) Central African Republic, and (iii) Gabon and
adjacent Congo. There is also evidence of limited haplotype admixture in northeastern Gabon
and southeast Cameroon. The phylogeographical patterns are broadly consistent with those
predicted by current Pleistocene refuge hypotheses for the region and suggest that historical
events have played an important role in shaping the population structure of this subspecies.
Keywords: control region, Gorilla gorilla, mitochondria, phylogeography
Received 25 August 2003; revision received 11 December 2003; accepted 6 January 2004

Introduction
Cyclical climate fluctuations during the late Quaternary
are believed to have had a considerable impact on the past
distribution and range dynamics of many African taxa
(Lanfranchi & Schwartz 1990; Maley 1996). The colder, drier
periods experienced during glacial maxima are believed to
have led to the retraction of tropical forests into refugia and
fostered allopatric divergence between isolated populations
(Haffer 1982; Kingdon 1990; Grubb 2001). Major montane
Correspondence: Stephen L. Clifford. Fax: + 241 67 72 95; E-mail:
stephen1clifford@yahoo.com
Stephen Clifford and Nicola Anthony contributed equally to this work.
2004 Blackwell Publishing Ltd

refugia have been identified in Cameroon and the highlands

associated with the western rift of eastern Democratic
Republic of Congo, Rwanda and Uganda ( Maley 1996;
Haffer 1982; Kingdon 1990; Grubb 2001). Additional minor
refugia have also been proposed, based on palynological,
geological and biogeographical data (Sosef 1994; Rietkirk
et al. 1995; Maley 1996; Grubb 2001). It has also been suggested that fluvial refugia may have existed along major
watercourses in the Congo basin where forest cover persisted
during the arid phases of the Pleistocene (Colyn 1991;
Grubb 2001). Several predictions have been made about
the genetic and evolutionary consequences of ice-age refugia
(Haffer 1982; Hewitt 1996; Dynesius & Jansson 2000) and
whilst molecular phylogeography has been used to test

1552 S . L . C L I F F O R D E T A L .
some of these predictions, most studies have focused on
temperate or arctic systems (e.g. Byun et al. 1997; Petit et al.
1997; Avise & Walker 1998; Avise et al. 1998; Arbogast 1999;
Holder et al. 1999). In contrast, surprisingly few phylogeographical studies have been carried out in equatorial Africa
with some notable exceptions (e.g. Morin et al. 1994; Goldberg
& Ruvolo 1997a,b; Gonder et al. 1997; Smith et al. 2000;
Flagstad et al. 2001; Jensen-Seaman & Kidd 2001; MulokoNtoutoume et al. 2001; Eggert et al. 2002).
Gorillas are restricted to closed canopy forest and
high-altitude montane forest and do not occur in forest
savannah mosaic habitats (Tutin et al. 1997). Thus, changes
in forest cover during Pleistocene glaciations may have had
a profound effect on the colonization patterns and regional
distribution of gorillas. Based on cranial and dental morphology, three subspecies of gorilla have traditionally been
recognized (Groves 1967, 1970), Gorilla gorilla gorilla (western lowland gorilla), G. g. graueri (eastern lowland gorilla)
and G. g. beringei (mountain gorilla). Western lowland gorillas
are the most numerous (Harcourt 1996) and are separated from eastern subspecies by more than 850 km. They
are found in Gabon, Cameroon, Equatorial Guinea, Congo,
Central African Republic (CAR), Cabinda and Nigeria
(Tutin & Vedder 2001). Eastern lowland gorillas occur in
fragmented populations of both lowland and highland
habitat in the Democratic Republic of Congo (DRC) (Hall
et al. 1998; Omari et al. 1999) whereas mountain gorillas
persist in two small populations in Rwanda and Uganda
(Harcourt 1996). A recent evaluation of the available data
has lead to a reclassification of the gorilla into two species
(Groves 2001), the western gorilla G. gorilla and the eastern
gorilla G. beringei. Within western gorillas, two subspecies
have been proposed: G. g. gorilla (western gorillas except those
in the Cross River area between Nigeria and Cameroon)
and G. g. diehli, comprising of no more than a few hundred
individuals in and around the Cross River. Within eastern
gorillas, three subspecies have been proposed: G. b. graueri
(eastern lowland), G. b. beringei (Virunga mountain gorilla)
and an as yet unnamed third taxonomic unit from the Bwindi
forest, Uganda (Sarmiento et al. 1996; Groves 2001).
Although gorillas exhibit substantial ecological and
morphological differentiation (Groves 1967, 1970, 2001;
Sarmiento et al. 1996; Doran & McNeilage 1998; Sarmiento
& Oates 2000; and references therein), molecular studies
have only recently begun to quantify levels of genetic variation within and between wild gorilla populations (Garner
& Ryder 1996; Field et al. 1998; Saltonstall et al. 1998; Clifford
et al. 1999, 2003; Jensen-Seaman & Kidd 2001; Oates et al.
2002). Information from mitochondrial DNA (mtDNA)
sequences has revealed high levels of variability within
western lowland gorillas (Ruvolo et al. 1994; Garner &
Ryder 1996; Noda et al. 2001), greater than that observed
within eastern or mountain gorillas (Garner & Ryder 1996).
However, very little information is currently available on

the geographical distribution of genetic variation in gorilla

populations of known origin. Several studies point to evidence of strong phylogeographical structure within gorillas.
Jensen-Seaman & Kidd (2001) have examined mtDNA
variation in several eastern lowland and mountain gorilla
populations throughout eastern Africa and Oates et al.
(1999, 2002) postulated a genetically distinct population of
gorillas in the Cross River region corresponding to one of
four geographically defined demes identified by Groves
(1967). This study therefore aims to expand on an earlier study
(Clifford et al. 2003) and to explore the potential role that
Pleistocene refugia may have played in shaping gorilla population genetic structure. The first hyper-variable region
(HV1) of the mitochondrial genome was chosen for investigation since previous studies have shown this marker to
be well suited to intraspecific studies in great apes (e.g. Morin
et al. 1993, 1994; Garner & Ryder 1996; Goldberg & Ruvolo
1997a,b; Gonder et al. 1997; Gagneux et al. 1999, 2001).

Materials and methods

Sample collection and DNA extraction
Shed hair collected from night nests (Tutin et al. 1995) comprised
all of our samples except a single fecal sample collected in
Gabon and a single museum pelt specimen reportedly collected
in the 1930s from Belar, Cameroon. Samples throughout the
entire gorilla range were collected over a period of several
years through collaborations with researchers from existing
study sites. Hair samples were stored at room temperature
in a plastic container with silica gel. The fecal sample was
collected in RNAlater (Ambion, Austin, TX, USA) and subsequently stored at 20 C. Table 1 lists these contributors
and the populations sampled. Hair was extracted using either
a modification of the Chelex-100 protocol of Walsh et al.
(1991) or a method modified from the protocol described
by Vigilant (1999) based on proteinase K digestion of hair
roots in a polymerase chain reaction (PCR) compatible buffer.
DNA from the museum pelt was extracted using the QIAamp
DNA kit extraction protocol (Qiagen). Fecal DNA was
extracted using the Qiagen Stool Extraction Kit (Qiagen).
The CITES numbers for exportation and importation of all
hair extracts are 00174 2 and 16640/01, respectively.

Mitochondrial DNA amplification

As DNA extracted from noninvasive samples is highly
degraded, only short DNA fragments could be amplified
reliably (Clifford et al. 2003). Nested primers were designed
to amplify a 258-base-pair (bp) fragment of the mitochondrial HV1 using the first-round primers PDPF1 (5CACCATCAGCACCCAAAGCTAATAT-3) and PDPR2
(5-TTGTGCGGGATATTGATTTCACGGA-3) and secondround primers L91-115 and H402-27 (Garner & Ryder 1996).
2004 Blackwell Publishing Ltd, Molecular Ecology, 13, 15511565

P H Y L O G E O G R A P H Y O F W E S T E R N L O W L A N D G O R I L L A S 1553
Table 1 List of sample sites, country of origin, site code, site number, number of individuals analysed per site and collectors of samples from wild
gorilla populations examined in this study. *The sample from Rabi (site 7) was from feces. All other samples were derived from shed hair.
**CDP (site 11) is a captive individual from the Centre de Primatologie (CDP) at CIRMF, Gabon, who originated from central Gabon.
***Samples from Belar (site 22) and Uele (site 23) are from Museum specimens

Site

Country

Code

Site
no.

Bwindi
Karisoke
Kahuzi-Biega

Uganda
Rwanda
DRC

BWD
RWD
KBG

1
2
3

4
2
6

Tshiaberimu
Itombwe
Conquati
Rabi*
Petit Loango
Lop

DRC
DRC
Congo
Gabon
Gabon
Gabon

TSH
ITW
CQT
RAB
PLO
LOP

4
5
6
7
8
9

1
3
1
1
1
3

Lastourville
CDP**

Gabon
Gabon

LAS
CDP

10
11

1
1

Ipassa
Belinga
Itombe
Lossi
Bai Hokou

Gabon
Gabon
Gabon
Congo
CAR

IPS
BEL
ITO
LOS
CAR

12
13
14
15
16

2
2
1
1
8

Nouabal-Ndoki
Lobk
Dja
Afi Mts./Cross River
Mont Alen

Congo
Cameroon
Cameroon
Nigeria
Equatorial
Guinea
Cameroon
DRC

NDK
LBK
DJA
CRS
EQG

17
18
19
20
21

1
5
4
4
5

(WCS). Sequences from Genbank (L76749, L76752)

Sequences from Genbank (L76750, L76751)
D. Bonny, K.P. Kiswele (CNRS), I. Omari, C. Sikubwabo
(ICCN), L. White, J. Hall, I. Bila-Isia, H. Simons Morland,
E. Williamson, K. Saltonstall, A. Vedder, K. Freeman, B.
Curran (WCS) J. Yamagiwa (Kyoto). Sequences from Genbank
(L76771, L76772, L76773, AF187549)
Sequence from Genbank (AF50738)
I. Omari, F. Bengana ( ICCN); J. Hart ( WCS)
B. Goossens (UWC), A. Jamart (HELP)
S. Lahm (IRET)
J. Yamagiwa (Kyoto University)
C. Tutin, K. Abernethy, E. Dimoto, J.T. Dinkagadissi, R.
Parnell, P. Peignot, B. Fontaine (CIRMF), M.E. Rogers, L. White,
B. Voysey, K. McDonald, (Edinburgh), R. Ham (Stirling), J.G.
Emptaz-Collomb
Y. Mihindou (WCS-MIKE)
J. Wickings (CIRMF) (equivalent to sequence from Genbank
L76764)
S. Lahm, J. Okouyi (IRET)
S. Lahm (IRET). Sequence from Genbank (L76763)
P. Telfer (NYU)
M. Bermejo, G. Illera, F. Maisels (ECOFAC)
M. Goldsmith (Tufts University), L. White (WCS). Sequences
from Genbank (AY079508, AY079509, AY079510, L76761)
P. Walsh (WCS)
L. White, L. Usongo (WCS)
E. Williamson (ECOFAC), L. Usongo (WCS/ECOFAC)
K. McFarland, J. Oates (CUNY, USA). E. Nwufoh (CRNP)
M. Bermejo, G. Illera (ECOFAC)

CAM
UEL

22
23

1
1

M. Harman (Powell-Cotton Museum)

Sequences from Genbank (AJ422244)

Belar***
Uele***

No.
individuals

Cycle conditions for both first- and second-round PCR were

as follows: 94 C for 3 min followed by 50 cycles of
94 C for 30 s, 50 C for 30 s and 72 C for 30 s and a final
step of 72 C for 10 min. Both extraction blanks and
reaction blanks containing only PCR reagents were also
included in nested reactions to control for potential contamination. Extraction blanks or PCR blanks almost never
yielded products. The exception to this was the occasional
amplification of human DNA which could be readily identified
owing to a large deletion (70 bp) in the gorilla HV1 sequence
relative to human and chimpanzee sequences (see Garner &
Ryder 1996). For fecal DNA extracts, PCR products were
amplified over 35 cycles from 1 L DNA using the L91/
H402 primer combination and an annealing temperature
of 62 C. Second-round PCR reactions were conducted
using identical conditions. All reactions contained final
concentrations of 1.5 mm Mg2+, 1 buffer (Invitrogen),
2004 Blackwell Publishing Ltd, Molecular Ecology, 13, 15511565

Collectors

200 m dNTPs, 0.2 m of each primer and 0.5 units of Taq

polymerase in a 10-L volume PCR reaction.

DNA sequencing and data analysis

PCR products were purified using the Turbo-purification
KitIII (Gene Clean) and were either cloned into the PCR2.1TOPO vector (Invitrogen) prior to sequencing with M13
Forward (20) and M13 reverse primers or were directly
sequenced with L91-115 and H402-427 primers. In all cases,
DNA was sequenced using the Big Dye V2.0 sequencing
kit (Applied Biosystems). Sequences derived from cloned
PCR products were assembled from a consensus of a mean
of 4.7 clones per PCR product. Several published haplotypes
of known geographical provenance were also included
in phylogenetic analyses. These sequences are available in
GenBank under the following accession numbers: L76749,

1554 S . L . C L I F F O R D E T A L .
AY530102 to AY530154). To test whether different PCR
primers yielded equivalent product and subsequent sequences,
a subset of seven samples (EQG1, EQG2, DJA2, CAR3,
CAR4, LOP1, LOP2) were PCR amplified using the primers
and conditions described by Oates et al. (2002) and the resulting sequences were found to match those generated using
the nested PCR strategy described above. In addition, a previous study (Clifford et al. 2003) based on a smaller fragment
of the mitochondrial control region, again used different
primer sets, and also yielded equivalent sequences.
Sequences were aligned in clustal X (Thompson et al.
1997) using the multiple alignment default parameters. A
26-bp region encompassing the poly C domain of HV1 was
excluded from principal phylogenetic analyses because of
difficulties in alignment and possible length and site heteroplasmy within this region (Bendall & Sykes 1995; Garner
& Ryder 1996). However, phylogenetic analysis motifs within
the poly C domain and diagnostic sites in the sequence
flanking the poly C domain were used to classify putative
Numt sequences into two categories (I and II). Table 2

L76750, L76751, L76752 (mountain gorillas); L76771, L76772,

L76773, AF187549, AF050738 (eastern lowland gorillas);
L76754, L76760, L76761, L76763, L76764, L76766, AY079508,
AY079509, AYO79510, AF250888 (western lowland gorillas).
Also included was the sequence (AJ422244) obtained from
a museum specimen from Bondo, in the Uele Valley
(D.R.C.) where gorillas do not occur (Hofreiter et al. 2003).
A series of candidate nuclear copies of mtDNA (Numts)
of the mtDNA control region available through GenBank
(Accession Numbers AF240448 AF240453, AF240455
AF240458) were also included in phylogenetic analyses.
Minor differences in the polycytosine (poly C) domain
sequence were observed between clones and were coded
by consensus. Occasionally base conflicts between plasmid
clones outside the poly C domain were encountered and
these changes were coded by majority. These base conflicts
could arise through (i) PCR errors, (ii) heteroplasmy which
has been diagnosed in hairs (Grzybowski 2000), or (iii) Numt
allele polymorphisms. The sequences generated from this
study have been deposited in GenBank (Accession Numbers

Table 2 Shared derived characters used to diagnose different Numt groups and consensus sequences from a 26 bp tract of the poly cytosine
(Poly C) domain within HV1. The base pair position of each diagnostic sites and the start and end sites of the Poly C sequence are derived
from a 258 bp alignment of all the sequences used in this study. Variable sites are coded by their respective degenerate code. Indels are
represented by consensus. The symbol (-) indicates a deletion relative to other sequences in the full alignment. The symbol (?) indicates
missing data. Sequence origin (Cloned, PCR product or Genbank) and haplogroup association/Numt class are also indicated

Sequence

0
7
9

0
8
9

0
9
2

0
9
3

1
1
2

1
3
8

1
5
7

1
6
3

Origin

RWD1_L76751
RWD2_L76750
BWD1
BWD2
BWD3_L76749
BWD4_L76752

C
C
C
C
C
C

C
C
C
C
C
C

C
C
C
C
C
C

C
C
C
C
C
C

CCCCTCACCCCCCATTCCCTGCTCAC
CCCCTCACCCCCCATTCCCTGCTCAC
CCCCTCACCCCCCATTCCCTGCTCAC
CCCCTCACCCCCCATTCCCTGCTCAC
CCCCTCACCCCCCATTCCCTGCTCAC
CCCCTCACCCCCCATTCCCTGCTCAC

A
A
A
A
A
A

T
T
T
T
T
T

GENBANK
GENBANK
PCR
PCR
GENBANK
GENBANK

TSH1_AF50738
KBG1
KBG2
KBG3_L76773
KBG4_AF187549
KBG5_L76771
KBG6_L76772
ITW1
ITW2
ITW3

C
C
C
C
C
C
C
C
C
C

C
C
C
C
C
C
C
C
C
C

T
T
T
T
T
T
T
T
T
T

C
C
C
C
C
C
C
C
C
C

CCCCTCACCCCCCATCCCTTGCCCAC
CCCCTCACCCCCCATCCCTTGCCCAC
CCCCTCACCCCCCATCCCTTGCCCAC
CCCCTCACCCCCCATCCCTTGCCCAC
CCCCTCACCCCC-ATCCCTTGCCCAC
CCCCTCACCCCC-ATCCCTTGCCCAC
CCCCTCACCCCC-ATCCCTTGCCCAC
CCCCTCACCCCC-ATCCCTTGCCCAC
CYCCTCACCCTC-ATCCCTTGCCCAC
CCCCTCACCCCC-ATCCCTTGCCCAC

A
A
A
A
A
A
A
A
A
A

C
C
C
C
C
C
C
C
C
C

GENBANK
PCR
PCR
GENBANK
GENBANK
GENBANK
GENBANK
PCR
PCR
PCR

CRS1
CRS4
CRS2
CRS3
LBK1
LBK2
LBK4
LBK5
DJA1
DJA4
DJA2
UEL1_AJ422244
IPS2
DJA3

C
C
C
C
C
C
C
C
C
C
C
C
C
C

C
C
C
C
C
C
C
C
C
C
C
C
C
C

T
T
T
T
T
T
T
T
T
T
C
C
C
C

C
C
C
C
C
C
C
C
C
C
C
C
C
C

CTCCC-CTTCCCCCCCCC-TCCTCCA
CCCCCTCTTCCCCCCCCC-TCCTCCA
CCCCCCCTTCCCCCCCCC-TCCTCCA
CCCCCCCTTCCCCCCCCC-TCCTCTA
YCCCYCCTTCCCCCCYCCCTTCTCCA
CCCCYCCTTCCCCYCYCC-TCCTCCA
CCCCCCCTTCCCCCCCCC-TTCTCCA
CTCCCCCTTCCCCCCCCC-TCCTCCA
CCCCCYCYTYCCCCCCCCCTCYTCYA
CCCCCCCTTCTCCCCCCC-TCCTCCA
CCCCCC-TT---------------CA
YYYYYYYYYYYYYYYYYYYYYYYYYA
CCCCCCCTT--CCCCCCCCCCCTTCA
CCCCCYCTT-CCCCTCCCCCCCTCCA

A
A
A
A
A
A
A
A
A
A
A
A
A
A

T
T
T
T
T
T
T
T
T
T
T
T
T
T

PCR
PCR
PCR
CLONE
CLONE
CLONE
PCR
CLONE
CLONE
CLONE
PCR
GENBANK
CLONE
CLONE

Poly C

Haplogroup/
Numt class

Haplogroup
A

Haplogroup
B

5
4
4
6
4
4
7

Haplogroup
C

2004 Blackwell Publishing Ltd, Molecular Ecology, 13, 15511565

P H Y L O G E O G R A P H Y O F W E S T E R N L O W L A N D G O R I L L A S 1555
Table 2 Continued

Sequence

0
7
9

0
8
9

0
9
2

0
9
3

1
1
2

1
3
8

1
5
7

1
6
3

Origin

CAR1
NDK1
CAR7
CAR8_AY079509
CAR3
CAR2_L76761
CAM1
CAR4
CAR6_AY079510
CAR5_AY079508
LBK3
EQG1
EQG2
EQG3
EQG5
EQG4
RAB1
LOS1
LAS1
BEL1
LOP3
ITO1
BEL2_L76763
LOP2
CQT1
CDP1_L76764
IPS1
PLO1
LOP1

C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C

C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C

T
T
T
T
T
T
T
T
T
T
T
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
T

C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C

CCCCCCCTTCCCCCCCCCCCGCACTG
CCCCCCCTTCCYCCCCCC-CGCACTG
CCCCCCCTTTCCCCCCCCCCGCACTG
CCCCCCCTTCNCCCCCCC-CGCACTG
CCCCCCCTTCCCCCCCCC-CGCACTG
CCCCCCCTTCCCCCCCCCCCGCACTG
CCCCCCCTTCCCCCCCCC-CGCACTG
CCCCCCCTTCCCCCCCCCCCGCACTG
CCCCCCCTTCCCCCCCCC-CGCATTG
CCCCCCCTTCNCCCCCCC-CGCACTG
CCCCCCCTTCCCCCCCCCCTGCACTG
CCCCCCCTTCCCCCTCCCCTCCTCTG
CCCCCCCTTCCCCCTCCCCTCCTCTG
YCCCCCCTTCCCCCCCCC-TCCTCTG
CCCCCCCTTCCCCCCCCC-TCCTCTG
CCCCCCCTTCCCCCCCCC-CGCTCTG
CCCCCCCTTCCCCCCCCCCCGCTCTG
CCCCCCCTTCCCCCCCCCCCGCTCTG
CCCCCCCTT-CCCCCYCC-CCYTCTG
CCCCCCCTTCCCCCCCCC--GCTCTG
CCCCCCCTTCCCCCCTCC-CGCTCTG
CCCCCCCTTNCCCCCCCC-CGCTCTG
CCCCCC-TTCCCCCCCCC-CGCTCTG
CCCCCCCTTCCCCCCCCC-CGCTCTG
CCCCCCCTTCCCCCCCCCC-GCTCTG
CCCCCCCTTCCCCCCCCC-CGCTCTG
CCCCCCCTTCCCCCCCCC-CGCTCTG
CCCCCCYTTCCYCCCCCC-CGYTYTG
CCCCCCCTTCCCCCCCCC-CGCTCTG

A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
?
A
A
A
A
A
A
A
A
A
A
A
A

T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T

CLONE
CLONE
PCR
GENBANK
PCR
GENBANK
PCR
PCR
GENBANK
GENBANK
PCR
PCR
PCR
CLONE
PCR
PCR
CLONE
CLONE
CLONE
CLONE
CLONE
CLONE
GENBANK
PCR
CLONE
GENBANK
CLONE
CLONE
PCR

ITO1a
AF240448
AF240456
BEL1a
L76760

C
T
T
T
T

C
C
C
C
T

C
C
C
C
C

C
C
C
C
C

CCCCCCCCT--C-----ACTGCTCCA
CCCCCCCCT--C-----ACTGCTCCA
CCCCCCCCT--C-----ACTGCTCCA
CCCCCCCCT--C-----ACTGCTCCA
CCCCCCCTC--CCCCCTACTGCTCCA

A
A
A
A
A

T
T
T
T
T

CLONE
GENBANK
GENBANK
CLONE
GENBANK

AF240452
ITW4
AF240449
AF240457
L76754
DJA5b

T
T
T
T
T
T

T
T
T
T
T
T

A
A
A
A
A
A

C
C
C
C
C
C

TTCCCC----CCCTCCGC----TCCA
TTCCCC----CCCTCCGC----TCCA
TTCCCC----CCCTCCGC----TCCA
TTCCCC----CCCTCCGC----TCCA
TTCCCC----CCCCCCCC--GCTCCA
TTCCCC----CCCCCCCCCSGYTCCA

G
G
G
G
G
G

G
G
G
G
G
G

GENBANK
PCR
GENBANK
GENBANK
GENBANK
CLONE

LBK5a
Rab1a
L76766
AF240455
AF240453
AF250888
AF240451

T
T
T
T
T
T
T

C
C
C
C
C
C
C

A
A
A
A
A
A
A

T
T
T
T
T
T
T

CCCCCC-----CCATCCCCTGCTCTA
CCCCCC-----CCATCCCCTGCTCTA
CCCCCC-----CCATCCCCTGCTCTA
CCCCCC-----CCATCCCCTGCTCTA
CCCCCC-----CCATCCCCTGCTCTA
CCCCCC-----CCATCCCCTGCTCTA
CCCCCC-----CCATCCCCTGCTCTA

G
G
G
G
G
G
G

CLONE
CLONE
GENBANK
GENBANK
GENBANK
GENBANK
GENBANK

DJA4a
ITW5
LOP4
AF240450
AF240458
DJA5a

T
T
T
T
T
T

C
C
C
C
C
C

A
A
N
A
A
A

C
C
C
C
C
C

ACCCCCTC--CCCACTTCCTGCTCCA
ACCCCCTC--CCCACTTCCTGCTCCA
ACCCCCTC--CCCAYTTNCTGTTCCA
ACCCCCTC--CCCACTTCCTGCTCCA
ACCCCCTC--CCCACTTCCTGCTCCA
ACCCCCTC--CCCACTTCCTGCTCCA

G
G
G
G
G
G

CLONE
PCR
PCR
GENBANK
GENBANK
CLONE

Poly C

2004 Blackwell Publishing Ltd, Molecular Ecology, 13, 15511565

Haplogroup/
Numt class

5
4
4
4
4
4
4
6 Haplogroup
D
4
4
4
4
4
4
7

Numt
Class I

5
4
4
4
4
6
4
4
4
4
7

Numt
Class II

1556 S . L . C L I F F O R D E T A L .
lists the poly C sequence and synapomorphic sites for each
sequence. These Numt sequences were included in initial
phylogenetic analyses but were removed from the minimum spanning network and subsequent population
genetic analyses. Putative Numts were identified in both
eastern and western gorilla samples from the following
sites: two samples from Itombwe, DRC (site 5), two samples from Dja (site 19), one sample from Lobk (site 18),
one sample from Rabi, Southwestern Gabon (site 7), one
sample from Lop, Central Gabon (site 9), and one sample
from Belinga (site 13) and Itombwe (site 14), both in Northeastern Gabon.
For phylogenetic analyses, the two data sets (with and
without Numts) were analysed with modeltest 3.04 (Posada
& Crandall 1998) to determine the substitution model that
best fitted the data according to a hierarchical likelihood
ratio test. The K81uf + G model was selected in both cases.
modeltest was also used to estimate among-site-rate heterogeneity by estimating the value of the shape parameter
of the gamma distribution. Estimates were derived separately for the complete data set including all putative Numt
sequences ( = 0.39) as well as for unique mitochondrial
haplotypes ( = 0.49). Sequences were then analysed by
the neighbour-joining (Saitou & Nei 1987) and maximum likelihood (Cavalli-Sforza & Edwards 1967) methods
implemented in paup 4.0b10 (Swofford 1998) using the
appropriate model and empirical base frequencies. The
programme arlequin version 2.0 (Schneider et al. 2000) was
used to estimate nucleotide diversity within nominal subspecies (Nei 1987), calculate per cent sequence divergence
estimates between mtDNA haplogroups, carry out a hierarchical analysis of molecular variance (amova: Excoffier
et al. 1992) and construct a minimum spanning network of
mtDNA haplotypes. For the amova, sequences were partitioned either by subspecies (sensu Groves 1970) or by the
four principal haplogroups (AD) recovered in our phylogenetic analysis. Populations within these major subdivisions were defined by their respective subgroups (C1, C2,
D13). The demographic parameter was estimated for
haplogroups and subgroups within haplogroups with sample
sizes of 10 sequences or more using a general nonlinear least
squares approach (Schneider & Excoffier 1999). Confidence
intervals for these estimated parameters ( = 0.05) and the
validity of a model of sudden demographic expansion within
haplogroups was also assessed (Schneider & Excoffier 1999).

Results
Sequence divergence patterns and population genetic
structure
In total, 83 sequences were analysed, 53 of which were
generated in this study and 30 were retrieved from GenBank. All sequences were examined over a 232-bp region

equivalent to the HV1 domain that excluded a 26-bp region

of the Poly C domain. Of these sequences, 59 of the 83
sequences were presumed mitochondrial (Fig. 1), of which
16 were derived from eastern gorillas (six mountain and
10 eastern lowland) and 43 from western lowland gorillas.
Two of these western lowland gorilla mitochondrial sequences
were amplified from museum specimens (CAM1 and UEL1).
Of the 43 western gorilla sequences, 20 were derived from
cloned PCR products, 16 were derived from direct sequencing of PCR products and seven were derived from GenBank.
Overall, 36 unique haplotypes (Fig. 2) were identified across
23 sites throughout the range of western lowland gorillas.
The number of individuals sequenced per site is illustrated in
Table 1. The remaining 24 sequences, comprising seven cloned,
three PCR and 14 GenBank derived sequences were characterized as Numts and following phylogenetic classification
were subsequently removed from population genetic analysis.
Phylogenetic analysis, using both neighbour-joining and
maximum likelihood methods gave concordant tree topologies with or without Numts sequences. Figure 1 shows
the neighbour-joining tree generated with all sequences
(n = 83) included in the analysis and Fig. 2 the maximum
likelihood tree with only unique mitochondrial haplotypes
(n = 36). Both phylogenetic analyses (Figs 1 and 2) and
a minimum spanning network of the data (Fig. 3a) recovered four principal haplogroups (AD). Haplogroups
A and B correspond to mountain and eastern lowland
gorillas, respectively. Haplogroups C and D are restricted
to western lowland gorillas, are largely nonoverlapping,
and are both distributed over wide geographical areas.
Figure 3(b) illustrates the spatial distribution of haplotypes
within the four major haplogroups and their proportional
representation. Haplogroup C (n = 14) spanned an area
from the Cross River in Nigeria (site 20), through Dja (site
19) to Lobk in Southeast Cameroon (site 18), Ipassa in
Gabon (site 12) and a museum sample collected from the
DRC (site 23). Haplogroup D (n = 29) comprised all other
western gorillas sites from the CAR, Congo, Equatorial
Guinea and Gabon (sites 618) and one museum sample
from southern Cameroon (site 22). Whereas haplogroup
C was the most diverse, there appeared to be little or no
geographical pattern among phylogenetic subdivisions
(C1, C2) within this group. Conversely, phylogenetic analysis of haplogroup D recovered three well-defined genetic
subdivisions (D1D3) that were each confined to the following areas: (i) Equatorial Guinea (D1), (ii) CAR (D2) and
(iii) Gabon and adjacent Congo (D3). Limited haplotype
mixing between major haplogroups C and D was evident
in southeastern Cameroon and northeastern Gabon where
one sample from Lobk, Cameroon (otherwise C) appeared
to be almost identical to haplotypes across the river in
CAR (D2). Similarly, one sample from northeastern Gabon
(otherwise D3) possessed a haplotype characteristic of
haplogroup C. Finally, a museum specimen ostensibly
2004 Blackwell Publishing Ltd, Molecular Ecology, 13, 15511565

P H Y L O G E O G R A P H Y O F W E S T E R N L O W L A N D G O R I L L A S 1557

Fig. 1 Neighbour-joining bootstrap consensus tree of gorilla mitochondrial HV1 sequences alongside candidate Numt sequences. Threeletter taxa name with number corresponds to sample identification given in Table 1 and GenBank accession numbers are given for those
samples retrieved from the database. Numbers above tree branches correspond to the percentage bootstrap replicates for that branch.
Estimates of bootstrap support are based on 1000 replicates and the tree is unrooted. Haplogroups A to D and subgroups C1, C2, D1, D2
and D3 are indicated, and candidate Numts (classes I and II) are shaded in grey.

2004 Blackwell Publishing Ltd, Molecular Ecology, 13, 15511565

1558 S . L . C L I F F O R D E T A L .
Fig. 2 Maximum likelihood tree with branch
lengths generated from unique mitochondrial HV1 haplotypes. Midpoint rooting
is employed. Three-letter taxa name with
number corresponds to sample identification
given in Table 1 and GenBank accession numbers are given for those samples retrieved
from the database. Taxa names labelled with
an asterisk represent multiple individuals
with shared common haplotype. These are
BWD2/3 (BWD2, BWD3_L76749), KBG1/3
(KBG1, KBG3_L76773), ITW1/2/3 (ITW1,
ITW2, ITW3), LBK1/2/5 (LBK1, LBK2,
LBK5), EQG1/2/3/5 (EQG1, EQG2, EQG3,
EQG5), CAR/LBK/NDK (CAR1, CAR7,
CAR8_AY079509, LBK3, NDK1) and GAB/
CON (BEL1, BEL2_L76763, CQT1, ITO1,
LOP2, LOP3, LAS1, LOS1, PLO1, RAB1).
Haplogroups A to D and subgroups C1,
C2, D1, D2 and D3 are indicated.

from southwestern Cameroon possessed a haplotype characteristic of gorillas from the Sangha river region.
Mean absolute pairwise sequence differences between
eastern (haplogroup A and B) and western lowland gorillas (haplogroups C and D) was high (37.79/232; 16.29%)
and recapitulates the large genetic distance previously
identified between eastern and western lowland gorillas.
By comparison, mean percentage pairwise estimates between mountain (haplogroup A) and eastern lowland
gorillas (haplogroup B) were relatively low (14.85/232;
6.40%). However, the most striking divergence was between haplogroups C and D within western gorillas which
at 21.59/232 (9.31%), is higher than the average divergence
between the two eastern subspecies. Within haplogroups
AD, pairwise divergences were lower in the eastern haplogroups A (1.93/232; 0.83%) and B (2.00/232; 0.86%), than
within the two western haplogroups, C (7.43/232; 3.20%)
and D (7.12/232; 3.07%). In addition, pairwise sequence
differences were lowest within subgroups from haplogroup D (and equivalent to values obtained for the eastern

subspecies), ranging from 0.59/232 (0.25%) in D1 to 2.00/

232 (0.86%) in D2 and 2.11/232 (0.91%) in D3. In contrast,
pairwise sequence differences were higher in the two subgroups C1 (3.24.43/232; 1.4%) and C2 (3.67/232; 1.6%).
Estimates of nucleotide diversity (Nei & Li 1979) were
also highest in western lowland gorillas overall (0.062
0.031) and were comparable with previous published estimates (Garner & Ryder 1996; Jensen-Seaman & Kidd 2001).
This value is over twice as large as that estimated for published human sequences (Vigilant et al. 1991) and approximately six times greater than that observed for either
eastern lowland (0.009 0.006) or mountain (0.008 0.006)
gorillas. Jensen-Seaman & Kidd (2001) also report similar
estimates of nucleotide diversity in their study of eastern
gorillas. Within western lowland gorillas, nucleotide diversity was equivalent in haplogroups C (0.032 0.018) and
D (0.031 0.017), and was equally distributed between
haplogroup C subdivisions C1 (0.014 0.009) and C2 (0.016
0.012), respectively, but not across subdivisions within
haplogroup D (0.003 0.003, 0.011 0.007 and 0.009 0.007
2004 Blackwell Publishing Ltd, Molecular Ecology, 13, 15511565

P H Y L O G E O G R A P H Y O F W E S T E R N L O W L A N D G O R I L L A S 1559
Fig. 3 (a) Minimum spanning network constructed using the programme arlequin
(Schneider et al. 2000) of pairwise absolute
differences between gorilla mitochondrial
DNA haplotypes. Three-letter taxa name with
number corresponds to sample identification
given in Table 1. The area of each circle is
proportional to the number of sequences in
each. Branch lengths are also proportional
and hash marks for closely related haplotypes indicate individual mutational steps.
Haplogroups A to D are colour coded and
subgroups C1, C2, D1, D2 and D3 are
indicated. (b) Geographic distribution and
haplogroup designation (AD) of sequences
sampled from sites 123. The area of each
circle is proportional to the number of
sequences analysed at each site. The present
day geographical distribution of gorillas is
shaded in grey. Within the western gorilla
range, subgroups within major halogroups
C and D are as follows, C1 (sites 1820), C2
(sites 12, 19, 23), D1 (site 21), D2 (sites 16 18,
22) and D3 (sites 615). Gorillas from site 12
( IPS Ipassa, Gabon) and 19 ( LBK Lobk,
Cameroon) exhibit haplotypes from both
major haplogroups (C and D) and this is
reflected in circle coloration.

in D1, D2 and D3, respectively). Of note are the values

obtained for subgroup D3 which not only covers a large
geographical area ( Fig. 3b) containing over 50% of extant
gorillas ( Harcourt 1996) but also possesses the lowest
genetic variability of any of the subgroups examined.
An amova, where genetic variation was partitioned
among the three traditional gorilla subspecies (mountain,
eastern lowland and western lowland) indicated that over
half of the total molecular variance (50.98%) was attributable to the differences among subspecies. A substantial
proportion of the total variance (43.47%) was also attributable
2004 Blackwell Publishing Ltd, Molecular Ecology, 13, 15511565

to differences among subdivisions (C1, C2, D1D3) within

haplogroups. When western gorillas were subdivided into
two groups corresponding to haplogroups C and D in our
phylogenetic analysis, the among group variance rose to
65.04% of the total molecular variance. In the western gorillas,
mismatch distribution patterns within haplogroups C
and D were bimodal and multimodal respectively, indicating
population structure and complex evolutionary histories
within these two haplogroups. In contrast, mismatch
distributions in the eastern haplogroup B were unimodal,
and were consistent with a model of sudden demographic

1560 S . L . C L I F F O R D E T A L .
expansion with a -value of 2.115, range 0.506 3.187. Haplogroups A and D1 had too few individuals to be included
in this analysis. When mismatch distributions within haplogroup D were analysed by subgroups with 10 or more
sequences (D2, D3), the pattern of pairwise differences
was also consistent with a model of sudden demographic
expansion with -values ranging from 2.699 (1.0815.56) in
subgroup D2, to 1.105 (0.000 3.202) in subgroup D3. These
values are comparable with previous estimates from eastern
lowland and mountain gorillas (Jensen-Seaman & Kidd 2001).

Characterization of putative Numt groups

Nuclear transfers of mitochondrial DNA copies have been
reported in both humans (Zischler et al. 1995; Mourier et al.
2001) and nonhuman primates (Collura & Stewart 1995;
van der Kuyl et al. 1995; Zischler et al. 1998; Mundy et al.
2000). Several presumed Numts of the gorilla HV1 region
were identified by phylogenetic analysis and are highlighted in grey boxes in the phylogeny depicted in Fig. 1.
The classification of sequences into one of two Numt
classes (labelled III) was based on synapomorphic sites
within HV1 and distinct motifs within the poly C (Table 2).
Numt class I was made up of divergent sequences from
two cloned PCR product sequences from western gorillas
(BEL1, ITO1) as well as two putative Numt sequences from
GenBank (AF240448, AF240456). Within Numt class II, five
cloned PCR products from four western gorillas (RAB1,
DJA4, DJA5 and LBK5) yielded highly divergent sequences
that clustered with putative Numt sequences (AF240449
AF240453, AF240455, AF240457, AF240458). In addition,
direct PCR sequencing of samples from two eastern (ITW4,
ITW5) and one western (LOP4) gorilla yielded only putative
Numt Class II sequences. Finally, four western lowland
gorilla sequences previously submitted to GenBank clustered
with sequences from either Numt Class I (L76760) or II
(L76754, L76766, AF250888).
It is possible that all of the sequences presented as western gorilla mitochondrial DNA in haplogroups C and D
(including mitochondrial sequences retrieved from GenBank) are Numts. This is a highly unlikely scenario, as
similar mitochondrial-like western gorilla control region
sequences have been amplified by independent research
groups (Horai et al. 1995; Garner & Ryder 1996; Xu & Arnason
1996; Oates et al. 2002; Clifford et al. 2003) using multiple
combinations of different primer pairs that all yielded
equivalent sequences. In addition, the two complete gorilla
mitochondrial genomes previously published by Horai
et al. (1995) and Xu & Arnason (1996) contain HV1 sequences
similar to the mitochondrial sequences published in this
paper, clustering strongly with haplogroups C and D,
respectively. The Xu & Arnason (1996) mitochondrial
genome, in particular, is highly unlikely to be of nuclear
origin because it was obtained from DNA enriched with

respect to mtDNA. Finally, our own analysis, involved

multiple lines of evidence including the separation of mitochondrial and putative Numts by PCR product cloning,
PCR amplification of HV1 using different PCR primer sets
and the identification of Numt group synapomorphic sites
and poly C motifs (see Table 2).

Phylogenetic relationships
Figure 1 depicts the phylogenetic relationships between
the 83 gorilla-derived sequences examined in this study
whereas Fig. 2 depicts the relationship between unique
gorilla mitochondrial haplotypes, excluding all Numts.
As reported previously (Ruvolo et al. 1994; Garner & Ryder
1996), phylogenetic analyses reveal a major evolutionary
split between eastern and western gorillas. Mountain
gorillas (haplogroup A) are clearly differentiated from all
eastern lowland gorillas (haplogroup B). The most striking
aspect of this analysis occurs in the western gorilla, with a
deep phylogenetic break apparent within western lowland
gorillas (haplogroups C and D) that is strongly supported
in both neighbour-joining and maximum likelihood analysis. The two haplogroups are predominantly monophyletic
with evidence of limited mtDNA exchange at locations
where the two haplogroups potentially come into contact.
This haplotype exchange is shown by the presence of
a Southeast Cameroon sequence (LBK3) in haplogroup D2
and a Gabonese sequence (IPS2) in haplogroup C2. Of note,
is a single museum specimen, collected in southwestern
Cameroon (CAM1) in the 1930s, that clusters with other
sequences from haplogroup D2, despite being more than
700 km west of any D2 gorilla examined in this study. A
second museum-derived sequence (AJ422244) from DRC,
where gorillas do not occur and which is closer to the
current eastern gorilla range, clusters in haplogroup C2
with sequences from Nigeria, Cameroon and Northern
Gabon. The unexpected placement of these two sequences
does however, raise questions about the reliability of using
museum specimens when examining phylogeography in
current populations (Hofreiter et al. 2003).

Discussion
Gorilla phylogeography
In Africa, the lower temperatures and greater aridity experienced during Pleistocene glacial maxima are thought
to have led to the fragmentation of rainforest taxa into
forest refugia (Maley 1996). Contraction of species ranges
into these refugia may have fostered divergence between
fragmented populations whereas rapid colonization during
periods of climate amelioration may have led to large
geographical areas being occupied by relatively few haplotypes. Genetic exchange between adjacent phylogroups
2004 Blackwell Publishing Ltd, Molecular Ecology, 13, 15511565

P H Y L O G E O G R A P H Y O F W E S T E R N L O W L A N D G O R I L L A S 1561
could result from zones of secondary contact between
expanding refugial populations. Genetic signatures of such
past historical events may be apparent in contemporary
population genetic structure and provide supporting evidence for the influence of such refugia on past population
genetic processes (Hewitt 1996; Jensen-Seaman & Kidd
2001). The most important finding of this study is the
exceptional geographical substructuring that exists within
western lowland gorillas. The large genetic distances between two major haplogroups within the western gorillas
is greater than the molecular divergence observed between
eastern gorilla subspecies and implies a relatively long
evolutionary separation. The results are broadly consistent
with previous morphological analysis (Groves 1967), where
western gorillas were portioned into four demes designated, Nigeria, Plateau, Coastal and Sangha. Two of these,
Nigeria and Plateau, coincide with the geographical distribution of haplogroup C in Nigeria and Cameroon, whereas
the distribution of subgroups D2 (CAR), D1 (Equatorial
Guinea) and D3 (Gabon and Congo) are largely equivalent
to the location of the Sangha (D2) and Coastal demes (D1,
D3), respectively. The geographical distribution of haplogroups C and D and their respective subgroups may therefore coincide, in part, with the locations of several major
forest refugia in Cameroon, Equatorial Guinea, Gabon and
Congo (Sosef 1994; Rietkirk et al. 1995; Maley 1996). All these
areas could have potentially harboured forest remnants
during dry phases of the Pleistocene and consequently led
to the allopatric separation and divergence of western
lowland gorillas. However, owing to the paucity of information on the precise location and nature of these refugia
(Livingstone 1982; White 2001), and the lack of other phylogeographical data covering this region, further sampling
and additional studies of other rain-forest-associated taxa
will help elucidate the relationship between Pleistocene
forest/fluvial refuges and gorilla genetic structure.
The haplotype diversity and bimodal mismatch distributions in haplogroup C, reflect a complex population
history and/or population genetic structure. Gorillas in
this haplogroup, despite being substantially less abundant
than their counterparts in haplogroup D are responsible
for over half of the nucleotide diversity found within western lowland gorillas. Previous morphological analysis
had placed the Cross River gorillas, from Nigeria (which
belong to subgroup C1), as the most distinct of the four
proposed demes (Groves 1967, 1970). However, haplotypes within this subgroup have a much wider geographical distribution and are similar to other haplotypes in
Cameroon and northeast Gabon. Further sampling in
haplogroup C is therefore required to elucidate the complex
patterns of genetic variation within this haplogroup more
clearly. In contrast, despite considerably greater sampling
within haplogroup D, the unimodal mismatch distributions and evidence of recent population expansion of two
2004 Blackwell Publishing Ltd, Molecular Ecology, 13, 15511565

out of the three subgroups (D2 and D3) are consistent with
a refugial expansion scenario (Hewitt 1996; Jensen-Seaman
& Kidd 2001). This is particularly true of subgroup D3,
where identical haplotypes are found across the entire
southern range of western gorillas in Gabon and Congo.
The present location of subgroup D2, restricted in southern
CAR and adjacent Congo, may be the result of a fluvial
refuge postulated to have persisted during the arid periods
of the Pleistocene (Colyn 1991). The identification of this
novel phylogeographical division in gorillas is also supported by previous morphological analyses (Groves 1967,
1970) that designate gorillas in this region as belonging to
the Sangha deme. The importance of fluvial refugia in
Africa, however, has yet to be evaluated. Overall, low levels
of mtDNA diversity exist within subgroups in haplogroup
D, the haplotype difference between subgroups being largely
responsible for the relatively higher levels of diversity
within this haplogroup as a whole.
Geographic boundaries of the distinct haplogroups do
not appear to coincide with any major rivers, suggesting
that present river courses do not constitute a major barrier
to gene flow in gorillas. The Sanaga river has previously
been reported as a taxonomic boundary for several groups,
including mandrills, drills, forest duikers and chimpanzee
subspecies (Gonder et al. 1997; Grubb 2001). This appears
not to be the case for gorillas, as populations on both sides
of the river belong to the same haplogroup. The apparent
phylogeographical break between adjacent haplogroups
C and D across the Sangha river may be simply coincident
with the expansion of two well-defined haplogroups. The
presence of a haplogroup D sequence in Lobk (LBK3)
suggests limited haplotype exchange between adjacent
groups C and D one side of the Sangha River. Similarly the
association of one of the Northern Gabon sequences from
Ipassa (IPS2) with sequences from Cameroon and Nigeria
suggest haplotype intermixing. Additional sampling in
potential zones of secondary contact should help elucidate
the geographical boundaries of these haplogroups more
clearly as well as quantify the degree of haplotype exchange
more accurately.
The existence of two distinct groups of presumed Numts
in the present study suggest that interpretation of mtDNA
phylogenies should proceed with caution. Incorporation
of Numts into mitochondrial analyses can potentially
confound interpretation of derived phylogenies and lead
to erroneous conclusions (Sorenson & Fleischer 1996;
Mourier et al. 2001). The presence of near identical eastern
and western lowland gorilla sequences within the putative
Numt clusters is consistent with the slower rates of molecular evolution in the nuclear genome (Brown et al. 1982)
and if homologous, suggest that these insertions may
have occurred prior to the divergence of eastern and
western gorillas. Alternatively, the presence of identical
alleles in eastern and western populations could be the

1562 S . L . C L I F F O R D E T A L .
result of recent gene flow or incomplete lineage sorting
between regional groups. We note that the identification
of several distinct Numt groups in this and other studies
(Garner & Ryder 1996; Jensen-Seaman & Kidd 2001)
suggests that multiple independent transpositions and/
or gene duplication events have occurred at different times
during gorilla history.

Conservation implications/conclusions
Phylogeographical analyses of intraspecific genetic variation can provide valuable information on how genetic
variation is partitioned within species and aid in the
identification of evolutionarily significant units (ESUs) for
conservation (Moritz 1994; 1995). The geographical distribution and almost complete reciprocal monophyly between
distinct haplogroups within western lowland gorillas
strongly supports the recognition of at least four distinct
evolutionary lineages comprising mountain, eastern lowland
and western lowland haplogroups C and D, although there
is evidence of limited gene flow between neighbouring
populations in Northeastern Gabon, Cameroon and the
Dzanga-Sangha region of CAR. This observation is not
inconsistent with genetic patterns predicted under Pleistocene refuge theory and does not preclude recognition of
haplogroups C and D as distinct evolutionary entities.
According to the model advanced by Moritz (1994), ESUs
are designated on the basis of reciprocal monophyly at
mitochondrial markers and significant divergence in
nuclear loci. Our result in this respect is then preliminary
as we present only data derived from maternal patterns of
genetic structure. Among recently diverged taxa, conflicts
between mitochondrial and nuclear loci (Shaw 2002) as
well as between loci in the nuclear genome (Machado &
Hey 2003) are to be expected. Therefore caution needs to
be exercised when defining evolutionary lineages derived
only from mitochondrial control region sequences. In
addition to conflicts between data sets, the criteria used to
establish significant evolutionary units remains contentious
(e.g. Crandall et al. 2000; Fraser & Bernatchez 2001; Moritz
2002; and references therein). Specifically, criticisms have
been made about (i) the over-reliance of molecular data in
identifying units for conservation, (ii) the fact that ESUs
do not necessarily represent adaptive units of significance
and (iii) that reciprocal monophyly is an overly restrictive
assumption given what is known of the gene dynamics of
speciation. Crandall et al. (2000) have called for a distinction
between historical and recent phenomena and proposed
that both ecological and genetic tests of nonexchangeability should be considered when defining ESUs. In
western gorilla populations, whilst there is evidence of
correspondence between patterns of genetic diversity in
the present study and morphological differences observed
between distinct population groups (cf. Groves 1967, 1970),

there is also evidence of disparity (Sarmiento et al. 1996;

Sarmiento & Oates 2000). For example, Nigerian gorillas
are morphologically distinct but belong to a large mitochondrial haplogroup that encompasses populations as far as
Southeastern Cameroon. One potential explanation for this
discrepancy is that morphological and ecological traits
under selection may evolve much more rapidly than the
neutral molecular markers used here so that disparities
may arise in recently diverged taxa. Moreover, regional
differences in potentially adaptive traits are difficult to
assess because of insufficient knowledge of the ecology and
behaviour of free-ranging gorilla populations. Nevertheless,
the mitochondrial data presented in this study significantly
advances our understanding of the molecular phylogeography of this endangered species of great ape and point to
regionally distinct mitochondrial lineages that reflect an
appreciable history of isolation from one another. Within
the context of the biogeography of this group, our results
also indicate that changes in the distribution of forest
vegetation during the Pleistocene could have fostered
divergence between regional populations and that conservation efforts should take these regional differences in
genetic diversity into account. Future studies should seek
to unequivocally identify nuclear insertions, assess genetic
structure using nuclear loci and collect further ecological
and behavioural data in order to better understand the
conservation status and evolutionary significance of the
major lineages identified in this study.

Acknowledgements
This work was funded by the Leverhulme Trust London and
the Darwin Initiative. We would like to thank all the collectors
listed in Table 1 for their sampling efforts, M. Jensen-Seaman
and B. Bradley for communicating data on Numts, A. J. Tosi and
P. Walsh for useful discussions and M. Kazmierczak for logistical
help.

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2004 Blackwell Publishing Ltd, Molecular Ecology, 13, 15511565

Stephen Clifford is a postdoctoral researcher based in Gabon

interested principally in primate population genetics and
conservation issues pertaining to the region. Nicola Anthony
is an assistant professor at University of New Orleans who is
interested in the molecular ecology and phylogeography of
tropical and temperate taxa. Jean Wickings is head of the
Molecular Ecology Unit (UGENET) at CIRMF, Gabon, specializing in aspects of African biodiversity and biogeography,
examining species ranging from primates and elephants to
tropical flora.