Mitochondrial DNA phylogeography of western lowland gorillas (Gorilla gorilla gorilla)

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  • 8/9/2019 Mitochondrial DNA phylogeography of western lowland gorillas (Gorilla gorilla gorilla)

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    Molecular Ecology (2004) 13, 1551–1565 doi: 10.1111/j.1365-294X.2004.02140.x

    © 2004 Blackwell Publishing Ltd

    BlackwellScience,Ltd

    Mitochondrial DNA phylogeography of western lowlandgorillas (Gorilla gorilla gorilla)

    STEPHEN L. CLIFFORD,*

     

    NICOLA M. ANTHONY,

     

     

    MIREILLE BAWE-JOHNSON,

     

    *

     

    KATE

    A. ABERNETHY,

     

    *‡

     

    CAROLINE E. G . TUTIN,

     

    *‡

     

    LEE J . T . WHITE,

     

    §

     

    MAGDELENA BERMEJO,

     

     

    MICHELLE L. GOLDSMITH,

     

    **

     

    KELLEY MCFARLAND,

     

    ††

     

    KATHRYN J . JEFFERY,

     

     

    MICHAEL

    W. BRUFORD

     

     

    and E. JEAN WI CKI NGS

     

    *

     

    *

     

    Centre International de Recherches Médicales, 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 evolu-tionary 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 thosepredicted 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

    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 sug-

    gested 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

     

    Correspondence: Stephen L. Clifford. Fax: +

     

    241 67 72 95; E-mail:

    [email protected]

    Stephen Clifford and Nicola Anthony contributed equally to this work.

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    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 phylogeo-

    graphical 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; Muloko-Ntoutoume 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 mor-

    phology, three subspecies of gorilla have traditionally been

    recognized (Groves 1967, 1970), Gorilla gorilla gorilla

     

    (west-

    ern 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 separ-

    ated 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 thosein 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 vari-ation 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 evid-

    ence 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 popu-

    lation genetic structure. The first hyper-variable region

    (HV1) of the mitochondrial genome was chosen for invest-

    igation 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 RNA

     

    later

     

    (Ambion, Austin, TX, USA) and sub-

    sequently stored at −

     

    20 °

     

    C. Table 1 lists these contributorsand 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 mito-

    chondrial HV1 using the first-round primers PDPF1 (5

     

     

    -

    CACCATCAGCACCCAAAGCTAATAT-3

     

     

    ) and PDPR2

    (5

     

     

    -TTGTGCGGGATATTGATTTCACGGA-3

     

     

    ) and second-

    round primers L91-115 and H402-27 (Garner & Ryder 1996).

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    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 con-

    tamination. Extraction blanks or PCR blanks almost never

    yielded products. The exception to this was the occasionalamplification 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 µ

     

    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 m

     

    m

     

    Mg

     

    2+

     

    , 1

     

    ×

     

     buffer (Invitrogen),

    200 µ

     

    m

     

    dNTPs, 0.2 µ

     

    m

     

    of each primer and 0.5 units of Taq

     

    polymerase in a 10-

     

    µ

     

    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 PCR2.1-

    TOPO vector (Invitrogen) prior to sequencing with M13Forward (

     

     

    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,

    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.

    No.

    individuals Collectors

    Bwindi Uganda BWD 1 4   (WCS). Sequences from Genbank (L76749, L76752)Karisoke Rwanda RWD 2 2 Sequences from Genbank (L76750, L76751)Kahuzi-Biega DRC KBG 3 6 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)Tshiaberimu DRC TSH 4 1 Sequence from Genbank (AF50738)Itombwe DRC ITW 5 3 I. Omari, F. Bengana ( ICCN); J. Hart (WCS)Conquati Congo CQT 6 1 B. Goossens (UWC), A. Jamart (HELP)Rabi* Gabon RAB 7 1 S. Lahm (IRET)Petit Loango Gabon PLO 8 1 J. Yamagiwa (Kyoto University)Lopé Gabon LOP 9 3 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-CollombLastourville Gabon LAS 10 1 Y. Mihindou (WCS-MIKE)CDP** Gabon CDP 11 1 J. Wickings (CIRMF) (equivalent to sequence from Genbank

    L76764)Ipassa Gabon IPS 12 2 S. Lahm, J. Okouyi (IRET)Belinga Gabon BEL 13 2 S. Lahm (IRET). Sequence from Genbank (L76763)Itombe Gabon ITO 14 1 P. Telfer (NYU)Lossi Congo LOS 15 1 M. Bermejo, G. Illera, F. Maisels (ECOFAC)Bai Hokou CAR CAR 16 8 M. Goldsmith (Tufts University), L. White (WCS). Sequences

    from Genbank (AY079508, AY079509, AY079510, L76761)Nouabalé-Ndoki Congo NDK 17 1 P. Walsh (WCS)Lobéké Cameroon LBK 18 5 L. White, L. Usongo (WCS)Dja Cameroon DJA 19 4 E. Williamson (ECOFAC), L. Usongo (WCS/ECOFAC)Afi Mts./Cross River Nigeria CRS 20 4 K. McFarland, J. Oates (CUNY, USA). E. Nwufoh (CRNP)Mont Alen Equatorial

    Guinea

    EQG 21 5 M. Bermejo, G. Illera (ECOFAC)

    Belar*** Cameroon CAM 22 1 M. Harman (Powell-Cotton Museum)Uele*** DRC UEL 23 1 Sequences from Genbank (AJ422244)

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    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 (

     

    Numt

     

    s)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

    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 result-

    ing sequences were found to match those generated using

    the nested PCR strategy described above. In addition, a previ-

    ous study (Clifford et al

     

    . 2003) based on a smaller fragmentof 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 hetero-

    plasmy 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

    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 derivedfrom a 258 bp alignment of all the sequences used in this study. Variable sites are coded by their respective degenerate code. Indels arerepresented by consensus. The symbol (-) indicates a deletion relative to other sequences in the full alignment. The symbol (?) indicatesmissing 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 Poly C

    1

    3

    8

    1

    5

    7

    1

    6

    3 Origin

    Haplogroup/

    Numt class

    RWD1_L76751   C C C C CCCCTCACCCCCCATTCCCTGCTCAC A T GENBANK

    Haplogroup

    A

    RWD2_L76750   C C C C CCCCTCACCCCCCATTCCCTGCTCAC A T GENBANKBWD1   C C C C CCCCTCACCCCCCATTCCCTGCTCAC A T PCR

    BWD2   C C C C CCCCTCACCCCCCATTCCCTGCTCAC A T PCRBWD3_L76749   C C C C CCCCTCACCCCCCATTCCCTGCTCAC A T GENBANKBWD4_L76752   C C C C CCCCTCACCCCCCATTCCCTGCTCAC A T GENBANK

    TSH1_AF50738   C C T C CCCCTCACCCCCCATCCCTTGCCCAC A C GENBANKKBG1   C C T C CCCCTCACCCCCCATCCCTTGCCCAC A C PCRKBG2   C C T C CCCCTCACCCCCCATCCCTTGCCCAC A C PCR HaplogroupKBG3_L76773   C C T C CCCCTCACCCCCCATCCCTTGCCCAC A C GENBANK BKBG4_AF187549   C C T C CCCCTCACCCCC-ATCCCTTGCCCAC A C GENBANKKBG5_L76771   C C T C CCCCTCACCCCC-ATCCCTTGCCCAC A C GENBANKKBG6_L76772   C C T C CCCCTCACCCCC-ATCCCTTGCCCAC A C GENBANKITW1   C C T C CCCCTCACCCCC-ATCCCTTGCCCAC A C PCRITW2   C C T C CYCCTCACCCTC-ATCCCTTGCCCAC A C PCRITW3   C C T C CCCCTCACCCCC-ATCCCTTGCCCAC A C PCR

    CRS1   C C T C CTCCC-CTTCCCCCCCCC-TCCTCCA A T PCR

    Haplogroup

    C

    CRS4   C C T C CCCCCTCTTCCCCCCCCC-TCCTCCA A T PCR

    CRS2   C C T C CCCCCCCTTCCCCCCCCC-TCCTCCA A T PCRCRS3   C C T C CCCCCCCTTCCCCCCCCC-TCCTCTA A T CLONELBK1   C C T C YCCCYCCTTCCCCCCYCCCTTCTCCA A T CLONELBK2   C C T C CCCCYCCTTCCCCYCYCC-TCCTCCA A T CLONELBK4   C C T C CCCCCCCTTCCCCCCCCC-TTCTCCA A T PCRLBK5   C C T C CTCCCCCTTCCCCCCCCC-TCCTCCA A T CLONEDJA1   C C T C CCCCCYCYTYCCCCCCCCCTCYTCYA A T CLONEDJA4   C C T C CCCCCCCTTCTCCCCCCC-TCCTCCA A T CLONEDJA2   C C C C CCCCCC-TT---------------CA A T PCRUEL1_AJ422244   C C C C YYYYYYYYYYYYYYYYYYYYYYYYYA A T GENBANKIPS2   C C C C CCCCCCCTT--CCCCCCCCCCCTTCA A T CLONEDJA3   C C C C CCCCCYCTT-CCCCTCCCCCCCTCCA A T CLONE

    5446447

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    Sequence

    0

    7

    9

    0

    8

    9

    0

    9

    2

    0

    9

    3

    1

    1

    2 Poly C

    1

    3

    8

    1

    5

    7

    1

    6

    3 Origin

    Haplogroup/

    Numt class

    CAR1   C C T C CCCCCCCTTCCCCCCCCCCCGCACTG A T CLONE

    Haplogroup

    D

    NDK1   C C T C CCCCCCCTTCCYCCCCCC-CGCACTG A T CLONE

    CAR7  C C T C CCCCCCCTTTCCCCCCCCCCGCACTG A T

    PCRCAR8_AY079509   C C T C CCCCCCCTTCNCCCCCCC-CGCACTG A T GENBANKCAR3   C C T C CCCCCCCTTCCCCCCCCC-CGCACTG A T PCRCAR2_L76761   C C T C CCCCCCCTTCCCCCCCCCCCGCACTG A T GENBANKCAM1   C C T C CCCCCCCTTCCCCCCCCC-CGCACTG A T PCRCAR4   C C T C CCCCCCCTTCCCCCCCCCCCGCACTG A T PCRCAR6_AY079510   C C T C CCCCCCCTTCCCCCCCCC-CGCATTG A T GENBANKCAR5_AY079508   C C T C CCCCCCCTTCNCCCCCCC-CGCACTG A T GENBANKLBK3   C C T C CCCCCCCTTCCCCCCCCCCTGCACTG A T PCREQG1   C C C C CCCCCCCTTCCCCCTCCCCTCCTCTG A T PCREQG2   C C C C CCCCCCCTTCCCCCTCCCCTCCTCTG A T PCREQG3   C C C C YCCCCCCTTCCCCCCCCC-TCCTCTG A T CLONEEQG5   C C C C CCCCCCCTTCCCCCCCCC-TCCTCTG A T PCREQG4   C C C C CCCCCCCTTCCCCCCCCC-CGCTCTG A T PCRRAB1   C C C C CCCCCCCTTCCCCCCCCCCCGCTCTG ? T CLONE

    LOS1   C C C C CCCCCCCTTCCCCCCCCCCCGCTCTG A T CLONELAS1   C C C C CCCCCCCTT-CCCCCYCC-CCYTCTG A T CLONEBEL1   C C C C CCCCCCCTTCCCCCCCCC--GCTCTG A T CLONELOP3   C C C C CCCCCCCTTCCCCCCTCC-CGCTCTG A T CLONEITO1   C C C C CCCCCCCTTNCCCCCCCC-CGCTCTG A T CLONEBEL2_L76763   C C C C CCCCCC-TTCCCCCCCCC-CGCTCTG A T GENBANKLOP2   C C C C CCCCCCCTTCCCCCCCCC-CGCTCTG A T PCRCQT1   C C C C CCCCCCCTTCCCCCCCCCC-GCTCTG A T CLONECDP1_L76764   C C C C CCCCCCCTTCCCCCCCCC-CGCTCTG A T GENBANKIPS1   C C C C CCCCCCCTTCCCCCCCCC-CGCTCTG A T CLONEPLO1   C C C C CCCCCCYTTCCYCCCCCC-CGYTYTG A T CLONELOP1   C C T C CCCCCCCTTCCCCCCCCC-CGCTCTG A T PCR

    ITO1a   C C C C CCCCCCCCT--C-----ACTGCTCCA A T CLONE

    Numt

    Class I

    AF240448   T C C C CCCCCCCCT--C-----ACTGCTCCA A T GENBANKAF240456   T C C C CCCCCCCCT--C-----ACTGCTCCA A T GENBANKBEL1a   T C C C CCCCCCCCT--C-----ACTGCTCCA A T CLONEL76760   T T C C CCCCCCCTC--CCCCCTACTGCTCCA A T GENBANK

    AF240452   T T A C TTCCCC----CCCTCCGC----TCCA G G GENBANK

    Numt

    Class II

    ITW4   T T A C TTCCCC----CCCTCCGC----TCCA G G PCRAF240449   T T A C TTCCCC----CCCTCCGC----TCCA G G GENBANKAF240457   T T A C TTCCCC----CCCTCCGC----TCCA G G GENBANKL76754   T T A C TTCCCC----CCCCCCCC--GCTCCA G G GENBANKDJA5b   T T A C TTCCCC----CCCCCCCCCSGYTCCA G G CLONE

    LBK5a   T C A T CCCCCC-----CCATCCCCTGCTCTA G - CLONERab1a   T C A T CCCCCC-----CCATCCCCTGCTCTA G - CLONEL76766   T C A T CCCCCC-----CCATCCCCTGCTCTA G - GENBANKAF240455   T C A T CCCCCC-----CCATCCCCTGCTCTA G - GENBANKAF240453   T C A T CCCCCC-----CCATCCCCTGCTCTA G - GENBANK

    AF250888  T C A T CCCCCC-----CCATCCCCTGCTCTA G -

    GENBANKAF240451   T C A T CCCCCC-----CCATCCCCTGCTCTA G - GENBANK

    DJA4a   T C A C ACCCCCTC--CCCACTTCCTGCTCCA G - CLONEITW5   T C A C ACCCCCTC--CCCACTTCCTGCTCCA G - PCRLOP4   T C N C ACCCCCTC--CCCAYTTNCTGTTCCA G - PCRAF240450   T C A C ACCCCCTC--CCCACTTCCTGCTCCA G - GENBANKAF240458   T C A C ACCCCCTC--CCCACTTCCTGCTCCA G - GENBANKDJA5a   T C A C ACCCCCTC--CCCACTTCCTGCTCCA G - CLONE

    Table 2 Continued

    5

    44444464

    444447

    544446

    44447

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    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 mini-

    mum 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 sam-

    ples from Dja (site 19), one sample from Lobéké (site 18),one sample from Rabi, Southwestern Gabon (site 7), one

    sample from Lopé, Central Gabon (site 9), and one sample

    from Belinga (site 13) and Itombwe (site 14), both in North-

    eastern Gabon.

    For phylogenetic analyses, the two data sets (with and

    withoutNumts) were analysed withmodeltest 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 hetero-

    geneity by estimating the value of the α shape parameter

    of the gamma distribution. Estimates were derived separ-

    ately for the complete data set including all putative Numt

    sequences (α = 0.39) as well as for unique mitochondrial

    haplotypes (α = 0.49). Sequences were then analysed by

    the neighbour-joining (Saitou & Nei 1987) and maxi-

    mum 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 sub-

    species (Nei 1987), calculate per cent sequence divergence

    estimates between mtDNA haplogroups, carry out a hier-

    archical analysis of molecular variance (amova: Excoffier

    et al. 1992) and construct a minimum spanning network of mtDNA haplotypes. For the amova, sequences were parti-

    tioned either by subspecies (sensu Groves 1970) or by the

    four principal haplogroups (A–D) recovered in our phylo-

    genetic analysis. Populations within these major subdivi-

    sions were defined by their respective subgroups (C1, C2,

    D1–3). The demographic parameter τ  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 (α = 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 geneticstructure

    In total, 83 sequences were analysed, 53 of which were

    generated in this study and 30 were retrieved from Gen-

    Bank. 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 fromcloned PCR products, 16 were derived from direct sequen-

    cing 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 charac-

    terized 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 topo-

    logies 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) re-

    covered four principal haplogroups (A–D). 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 areafrom the Cross River in Nigeria (site 20), through Dja (site

    19) to Lobéké 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 6–18) 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 ana-

    lysis of haplogroup D recovered three well-defined genetic

    subdivisions (D1–D3) that were each confined to the fol-lowing 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 Lobéké, 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

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    Fig. 1 Neighbour-joining bootstrap consensus tree of gorilla mitochondrial HV1 sequences alongside candidate Numt sequences. 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. 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.

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    from southwestern Cameroon possessed a haplotype char-

    acteristic of gorillas from the Sangha river region.

    Mean absolute pairwise sequence differences between

    eastern (haplogroup A and B) and western lowland goril-

    las (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 be-

    tween mountain (haplogroup A) and eastern lowland

    gorillas (haplogroup B) were relatively low (14.85/232;

    6.40%). However, the most striking divergence was be-tween 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

    A–D, pairwise divergences were lower in the eastern haplo-

    groups 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 haplo-

    group 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 sub-

    groups 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 esti-

    mates (Garner & Ryder 1996; Jensen-Seaman & Kidd 2001).

    This value is over twice as large as that estimated for pub-

    lished human sequences (Vigilant et al. 1991) and approx-

    imately six times greater than that observed for eithereastern 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 diver-

    sity 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

    Fig. 2 Maximum likelihood tree with branch

    lengths generated from unique mitochond-

    rial HV1 haplotypes. Midpoint rooting

    is employed. Three-letter taxa name with

    number corresponds to sample identification

    given in Table 1 and GenBank accession num-

     bers are given for those samples retrieved

    from the database. Taxa names labelled with

    an asterisk represent multiple individualswith 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.

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

    obtained for subgroup D3 which not only covers a largegeographical 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 attribut-

    able to the differences among subspecies. A substantial

    proportion of the total variance (43.47%) was also attributable

    to differences among subdivisions (C1, C2, D1–D3) within

    haplogroups. When western gorillas were subdivided intotwo 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

    Fig. 3 (a) Minimum spanning network con-

    structed 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 proportionaland hash marks for closely related haplo-

    types 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 (A–D) of sequences

    sampled from sites 1–23. 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 18–20), C2

    (sites 12, 19, 23), D1 (site 21), D2 (sites 16–18,22) and D3 (sites 6–15). Gorillas from site 12

    ( IPS — Ipassa, Gabon) and 19 (LBK — Lobéké,

    Cameroon) exhibit haplotypes from both

    major haplogroups (C and D) and this is

    reflected in circle coloration.

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    expansion with a τ-value of 2.115, range 0.506–3.187. Hap-

    logroups A and D1 had too few individuals to be included

    in this analysis. When mismatch distributions within hap-

    logroup 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 τ-values ranging from 2.699 (1.081–5.56) in

    subgroup D2, to 1.105 (0.000–3.202) in subgroup D3. Thesevalues 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 high-

    lighted in grey boxes in the phylogeny depicted in Fig. 1.

    The classification of sequences into one of two Numt

    classes (labelled I–II) 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 putativeNumt  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 west-

    ern gorilla mitochondrial DNA in haplogroups C and D

    (including mitochondrial sequences retrieved from Gen-

    Bank) 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 multiplecombinations 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 mito-

    chondrial 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 ana-

    lysis. 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 ex-

    perienced 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 haplo-

    types. Genetic exchange between adjacent phylogroups

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    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 evid-

    ence 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 withinwestern lowland gorillas. The large genetic distances be-

    tween 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’ design-

    ated, Nigeria, Plateau, Coastal and Sangha. Two of these,

    Nigeria and Plateau, coincide with the geographical dis-

    tribution 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 haplo-

    groups C and D and their respective subgroups may there-

    fore 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 informa-

    tion on the precise location and nature of these refugia

    (Livingstone 1982; White 2001), and the lack of other phylo-

    geographical data covering this region, further samplingand 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 distri-

     butions 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 west-

    ern lowland gorillas. Previous morphological analysis

    had placed the Cross River gorillas, from Nigeria (which

     belong to subgroup C1), as the most distinct of the fourproposed ‘demes’ (Groves 1967, 1970). However, haplo-

    types within this subgroup have a much wider geograph-

    ical 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 distribu-

    tions and evidence of recent population expansion of two

    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 periodsof the Pleistocene (Colyn 1991). The identification of this

    novel phylogeographical division in gorillas is also sup-

    ported 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 Lobéké (LBK3)

    suggests limited haplotype exchange between adjacent

    groups C and D one side of the Sangha River. Similarly theassociation 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 leadto 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 mole-

    cular 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

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    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 vari-

    ation 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 distribu-

    tion 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 Pleis-

    tocene 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) aswell 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 restrictiveassumption 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 nonexchange-

    ability 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 mitoch-

    ondrial 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 disparitiesmay 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 phylogeo-

    graphy 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 con-

    servation 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 andthe 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.

    References

    Arbogast BS (1999) Mitochondrial DNA phylogeography of the

    New World flying squirrels (Glaucomys): implications for Pleis-

    tocene biogeography. Journal of Mammology, 80, 142–155.

    Avise JC, Walker D (1998) Pleistocene phylogeographic effects on

    avian populations and the speciation process. Proceedings of theRoyal Society of London, Series B, 265, 457–463.

    Avise JC, Walker D, Johns GC (1998) Speciation durations and

    Pleistocene effects on vertebrate phylogeography.Proceedings of 

    the Royal Society of London, Series B, 265, 1707–1712.

    Bendall KE, Sykes BC (1995) Length heteroplasmy in the first

    hypervariable segment of the human mtDNA control region.

     American Journal of Human Genetics, 57, 248–256.

    Brown WM, Prager EM, Wang A, Wilson AC (1982) Mitochondrial

    DNA sequences of primates: tempo and mode of evolution.

     Journal of Molecular Evolution, 18, 225–239.

  • 8/9/2019 Mitochondrial DNA phylogeography of western lowland gorillas (Gorilla gorilla gorilla)

    13/15

    PHYLOGEOGRAPHY OF WESTERN LOWLAND GORILLAS 1563

    © 2004 Blackwell Publishing Ltd, Molecular Ecology, 13, 1551–1565

    Byun SA, Koop BF, Reimchen TE (1997) North American black

     bear mtDNA phylogeography: implications for morphology

    and the Haida Gwaii glacial refugium controversy. Evolution,

    51, 1647–1653.

    Cavalli-Sforza LL, Edwards AWF (1967) Phylogenetic analysis:

    models and estimation procedures. American Journal of Human

    Genetics, 19, 233–257.

    Clifford SL, Jeffery K, Bruford MW, Wickings EJ (1999) Identifica-

    tion of polymorphic microsatellite loci in the gorilla (Gorilla gorilla gorilla) using human primers: application to non-invasively

    collected hair samples. Molecular Ecology, 8, 1556–1558.

    Clifford SL, Abernethy K, White L, Tutin CEG, Bruford MW,

    Wickings EJ (2003) Genetic studies of western gorillas. In: Gorilla

    Biology: a Multidisciplinary Perspective (eds Taylor A, Goldsmith

    M), pp. 269–292. Cambridge University Press, Cambridge.

    Collura RV, Stewart C-B (1995) Insertions and duplications of 

    mtDNA in the nuclear genomes of Old World monkeys and

    hominoids. Nature, 378, 485–489.

    Colyn M (1991) L’importance zoogéographique du Bassin du fleuve

    Zaïre pour la spéciation. Annales Sciences Zoologiques, 264, 180–185.

    Crandall KA, Bininda-Emonds ORP, Mace GM, Wayne RK (2000)

    Considering evolutionary processes in conservation biology.

    Trends in Ecology and Evolution, 15, 290–295.Doran D, McNeilage A (1998) Gorilla ecology and behaviour.

    Evolutionary Anthropology, 6, 120–131.

    Dynesius M, Jansson R (2000) Evolutionary consequences of changes

    in species’ geographical distributions driven by Milankovitch

    climate oscillations. Proceedings of the National Academy of Sciences

    of the USA, 97, 9115–9120.

    Eggert SL, Rasner CA, Woodruff DS (2002) The evolution and

    phylogeography of the African elephant inferred from mito-

    chondrial DNA sequence and nuclear microsatellite markers.

    Proceedings of the Royal Society of London, Series B, 269, 1993–2006.

    Excoffier L, Smouse P, Quattro J (1992) Analysis of molecular vari-

    ance inferred from metric distances among DNA haplotypes:

    application to human mitochondrial DNA restriction data.

    Genetics, 131, 479–491.

    Field D, Chemnick L, Robbins M, Garner K, Ryder OA (1998)

    Paternity determination in captive lowland gorillas and orang

    utans and wild mountain gorillas by microsatellite analysis.

    Primates, 3, 199–209.

    Flagstad O, Syvertsen PO, Stenseth NC, Jakobsen KS (2001) En-

    vironmental change and rates of evolution: the phylogeographic

    pattern within the hartebeest complex as related to climatic

    variation. Proceedings of the Royal Society of London, Series B, 268,

    667–677.

    Fraser DJ, Bernatchez L (2001) Adaptive evolutionary conserva-

    tion: towards a unified concept for defining conservation units.

     Molecular Ecology, 10, 2741–2752.

    Gagneux P, Wills C, Gerloff U et al. (1999) Mitochondrial sequences

    show diverse evolutionary histories of African hominoids.

    Proceedings of the National Academy of Sciences of the USA , 96,

    5077–5082.

    Gagneux P, Gonder MK, Goldberg TL, Morin PA (2001) Gene flow

    in wild chimpanzee populations: what genetic data tell us about

    chimpanzee movement over space and time. Philosophical Trans-

    actions of the Royal Society of London, Series B, 356, 889–897.

    Garner KJ, Ryder OA (1996) Mitochondrial DNA diversity in

    gorillas. Molecular Phylogenetics and Evolution, 6, 39–48.

    Goldberg TL, Ruvolo M (1997a) Molecular phylogenetics and

    historical biogeography of east African chimpanzees. Biological

     Journal of the Linnean Society, 61, 301–324.

    Goldberg TL, Ruvolo M (1997b) The geographic apportionment of 

    mitochondrial genetic diversity in east African Chimpanzees,

    Pan troglodytes schweinfurthii. Molecular Biology and Evolution, 14,

    976–984.

    Gonder MK, Oates JF, Disotell TR, Forster MRJ, Morales JC,

    Melnick DJ (1997) A new west African chimpanzee subspecies?

    Nature, 388, 337.

    Groves CP (1967) Ecology and taxonomy of the gorilla. Nature,

    213, 890–893.Groves CP (1970) Population systematics of the gorilla. Journal of 

    Zoology, 161, 287–300.

    Groves CP (2001) Primate Taxonomy, p. 350. Smithsonian Institute,

    Washington DC.

    Grubb P (2001) Endemism in African rain forest mammals. In:

     African Rainforest Ecology and Conservation (eds Weber W, White

    LJT, Vedder A, Naughton-Treves L), pp. 88–100. Yale Univer-

    sity Press, New Haven and London.

    Grzybowski T (2000) Extremely high levels of human mitochon-

    drial DNA heteroplasmy in single hair roots. Electrophoresis, 21,

    548–553.

    Haffer J (1982) General aspects of the refuge theory. In: Biological

    Diversification in the Tropics  (ed. Prance GT), pp. 641–657.

    Columbia University Press, New York.Hall JS, Saltonstall K, Inogwabini B-I, Omari I (1998) Distribution,

    abundance and conservation status of Grauer’s gorilla.Oryx, 32,

    122–130.

    Harcourt A (1996) Is the gorilla a threatened species? How should

    we judge? Biological Conservation, 75, 165–176.

    Hewitt GM (1996) Some genetic consequences of ices ages, and

    their role in divergence and speciation. Biological Journal of the

    Linnean Society, 58, 27–276.

    Hofreiter M, Siedel H, Van Neer W, Vigilant L (2003) Mitochon-

    drial DNA sequence from an enigmatic gorilla population (Gorilla

     gorilla uellensis).  American Journal of Physical Anthropology, 121,

    361–368.

    Holder K, Montgomerie R, Friesen VL (1999) Glacial vicariance

    and historical biogeography of rock ptarmigan (Lagopus mutus)

    in the Bering region. Evolution, 53, 1936–1950.

    Horai S, Hayasaka K, Kondo R, Tsugane K, Takahata N (1995)

    Recent African origin of modern humans revealed by complete

    sequences of hominoid mitochondrial DNAs. Proceedings of the

    National Academy of Sciences of the USA , 92, 532–536.

     Jensen-Seaman MI, Kidd KK (2001) Mictochondrial DNA vari-

    ation and biogeography of eastern gorillas.  Molecular Ecology,

    10, 2241–2247.

    Kingdon J (1990) Island Africa. Collins, London.

    van der Kuyl AC, Kuiken CL, Dekker JT, Perizonius WRK,

    Goudsmit J (1995) Nuclear counterparts of the cytoplasmic

    mitochondrial 12S rRNA gene: a problem of ancient DNA and

    molecular phylogenies. Journal of Molecular Evolution, 40, 652–657.

    Lanfranchi R, Schwartz D (1990) Paysages Quaternaires de l’Afrique

    Centrale Atlantique. ORSTOM, Paris.

    Livingstone DA (1982) Quaternary geography of Africa and

    the refuge theory. In: Biological Diversification in the Tropics (ed.

    Prance GT), pp. 523 –535. Columbia University Press, New York.

    Machado CA, Hey J (2003) The causes of phylogenetic conflict in

    a classic Drosophila species group. Proceedings of the Royal Society

    of London Series B-Biological Sciences, 270, 1193–1202.

    Maley J (1996) The African rain-forest — main characteristics of 

    changes in vegetation and climate change from the Upper

    Cretaceous to the Quaternary. Proceedings of the Royal Society of 

    Edinburgh, Series B, 104, 31–73.

  • 8/9/2019 Mitochondrial DNA phylogeography of western lowland gorillas (Gorilla gorilla gorilla)

    14/15

    1564 S . L . C L I F F O R D E T A L .

    © 2004 Blackwell Publishing Ltd, Molecular Ecology, 13, 1551–1565

    Morin PA, Wallis J, Moore JJ, Chakraborthy R, Woodruff DS (1993)

    Non-invasive sampling and DNA amplification for paternity

    exclusion, community structure and phylogeography in wild

    chimpanzees. Primates, 34, 347–356.

    Morin PA, Moore JJ, Chakraborthy R, Jin L, Goodall J, Woodruff DS

    (1994) Kin selection, social structure, gene flow, and the evolu-

    tion of chimpanzees. Science, 265, 1193–1201.

    Moritz C (1994) Defining evolutionary significant units for conser-

    vation. Trends in Ecology and Evolution, 9, 373–376.Moritz C (1995) Uses of molecular phylogenies for conservation.

    Philosophical Transactions of the Royal Society of London, Series B,

    349, 113–118.

    Moritz C (2002) Strategies to protect biological diversity and the

    evolutionary processes that sustain it. Systematic Biology, 51,

    238–254.

    Mourier T, Hansen AJ, Willerslev E, Arctander P (2001) The

    human genome project reveals a continuous transfer of large

    mitochondrial fragments to the nucleus.  Molecular Biology and

    Evolution, 18, 1833–1837.

    Muloko-Ntoutoume N, Petit RM, White L, Abernethy K (2001)

    Chloroplast DNA variation in a rainforest tree ( Aucoumea klaine-

    ana, Burseraceae) in Gabon. Molecular Ecology, 9, 359–363.

    Mundy NI, Pissinatti A, Woodruff DS (2000) Multiple nuclearinsertions of mitochondrial cytochrome b sequences in callitrichine

    primates. Molecular Biology and Evolution, 17, 1075–1080.

    Nei M (1987)  Molecular Evolutionary Genetics. Columbia Univer-

    sity, New York.

    Nei M, Li W-H (1979) Mathematical model for studying genetic

    variation in terms of restriction endonucleases. Proceedings of the

    National Academy of Sciences of the USA, 76, 5269–5273.

    Noda R, Kim CG, Takenaka O et al.  (2001) Mitochondrial 16S

    rRNA sequence diversity of hominoids.  Journal of Heredity, 92,

    490–496.

    Oates JF, McFarland KL, Stumpf RM et al. (1999) New findings on

    the distinctive gorillas of the Nigeria–Cameroon border region.

     American Journal of Physical Anthropology, 28, 213–214.

    Oates JF, McFarland KL, Groves JL, Bergl RA, Linder JL, Disotell TR

    (2002) The Cross River gorilla: natural history and status of a

    neglected and critically endangered subspecies. In: Gorilla Bio-

    logy: a Multidisciplinary Perspective (eds Taylor A, Goldsmith M),

    pp. 472–502. Cambridge University Press, Cambridge.

    Omari I, Hart JA, Butynski TM (1999) The Itombwe Massif, Demo-

    cratic Republic of Congo: biological surveys and conservation,

    with an emphasis on Grauer’s gorilla and birds endemic to the

    Albertine Rift. Oryx, 33, 301–322.

    Petit R, Pineau E, Demesure B, Bacilieri R, Dacousso A, Kremer A

    (1997) Chloroplast DNA footprints of postglacial recolonization

     by oaks. Proceedings of the National Academy of Sciences of the USA,

    94, 9996–10001.

    Posada D, Crandall KH (1998) Modeltest: testing the model of 

    DNA substitution. Bioinformatics , 14, 817–818.

    Rietkirk M, Ketner P, De Wilde JJFE (1995) Caesalpinoideae and

    the study of forest refuges in central Africa. In: The Biodiversity

    of African Plants (eds van der Maesen LJG, van der Burgt XM,

    van Medenbach de Rooy JM), pp. 608–623. Kluwer. Academic

    Publishers, the Netherlands.

    Ruvolo M, Pan D, Zehr S, Goldberg T, Disotell TR, von Dornum M

    (1994) Gene trees and hominoid phylogeny. Proceedings of the

    National Academy of Sciences of the USA, 91, 8900–8904.

    Saitou N, Nei M (1987) The neighbor-joining method: a new

    method for reconstructing phylogenetic trees. Molecular Biology

    and Evolution, 14, 399–411.

    Saltonstall KG, Amato G, Powell J (1998) Mitochondrial DNA

    variability in Grauer’s gorillas of Kahuzi-Biega National Park.

     Journal of Heredity, 69, 129–135.

    Sarmiento EE, Oates JF (2000) The Cross River gorillas: a distinct

    subspecies, Gorilla gorilla diehli Matschie 1904. American Museum

    Novitates, XX, 3304.

    Sarmiento EE, Butynski TM, Kalina J (1996) Gorillas of Bwindi-

    Impenetrable forest and the Virunga volcanoes: taxonomic implica-

    tions of morphological and ecological differences.  American Journal of Primatology, 40, 1–21.

    Schneider S, Excoffier L (1999) Estimation of past demographic

    parameters from the distribution of pairwise differences when

    the mutation rates vary among sites: application to human

    mitochondrial DNA. Genetics, 152, 1079–1089.

    Schneider S, Roessli D, Excoffier L (2000)  Arlequin, a Software for

    Population Genetics Data Analysis, Version 2.000. Genetics and

    Biometry Laboratory, Department of Anthropology, University

    of Geneva, Geneva Switzerland.

    Shaw KL (2002) Conflict between nuclear and mitochondrial DNA

    phylogenies of a recent species radiation: what mtDNA reveals

    and conceals about modes of speciation in Hawaiian crickets.

    Proceedings of the National Academy of Sciences of the USA , 99,

    16122–16127.Smith TB, Holder K, Girman D et al.  (2000) Comparative avian

    phylogeography of Cameroon and Equatorial Guinea mountains:

    implications for conservation.  Molecular Ecology , 9, 1505–

    1516.

    Sorenson MD, Fleischer RC (1996) Multiple independent trans-

    positions of mitochondrial DNA control region sequences to the

    nucleus. Proceedings of the National Academy of Sciences of the

    USA, 93, 15239–15243.

    Sosef MSM (1994) Studies in Begoniaceae. Agricultural University

    Papers, Wageningen, the Netherlands.

    Swofford DL (1998) PAUP*, Phylogenetic Analysis Using Parsimony

    V4. Sinauer Associates, Sunderland MA.

    Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG

    (1997) The CLUSTAL_X windows interface: flexible strategies

    for multiple sequence alignment aided by quality analysis tools.

    Nucleic Acid Research, 25, 4876–4882.

    Tutin CEG, Vedder A (2001) Gorilla conservation and research

    in central Africa. In:  African Rainforest Ecology and Conservation

    (eds Weber W, White LJT, Vedder A, Naughton-Treves L),

    pp. 429–448. Yale University Press, New Haven and London.

    Tutin CEG, Parnell RJ, White LJT, Fernandez M (1995) Nest build-

    ing by lowland gorillas in the Lopé Reserve, Gabon: environ-

    mental influences and implications for censusing. International

     Journal of Primatology, 16, 53–76.

    Tutin CEG, White LJT, Mackanga-Missandzou A (1997) The

    use by rain forest mammals of natural forest fragments in

    an equatorial African savanna. Conservation Biology, 11, 1190–

    1203.

    Vigilant L (1999) An evaluation of techniques for the extraction

    and amplification of DNA from naturally shed hairs. Biological

    Chemistry, 380, 1329–1331.

    Vigilant L, Stoneking M, Harpending H, Hawkes K, Wilson AC

    (1991) African populations and the evolution of human mito-

    chondrial DNA. Science, 253, 1503–1507.

    Walsh PS, Metzger DA, Higuchi R (1991) Chelex 100 as a medium

    for simple extraction of DNA for PCR-based typing from forensic

    material. Biotechniques, 10, 506–513.

    White LJT (2001) The African rain forest. In:  African Rainforest

    Ecology and Conservation (eds Weber W, White LJT, Vedder A,

  • 8/9/2019 Mitochondrial DNA phylogeography of western lowland gorillas (Gorilla gorilla gorilla)

    15/15

    PHYLOGEOGRAPHY OF WESTERN LOWLAND GORILLAS 1565

    © 2004 Blackwell Publishing Ltd, Molecular Ecology, 13, 1551–1565

    Naughton-Treves L), pp. 1–29. Yale University Press, New

    Haven and London.

    Xu X, Arnason U (1996) A complete sequence of the mitochondrial

    genome of the western lowland gorilla.  Molecular Biology and

    Evolution, 13, 691–698.

    Zischler H, Geisert H, Castresana J (1998) A hominoid-specific

    nuclear insertion of the mitochondrial d-loop: implications for

    reconstructing ancestral mitochondrial sequences.  Molecular

    Biology and Evolution, 15, 463–469.Zischler H, Geisert H, von Haeseler A, Pääbo S (1995) A nuclear

    fossil of the mitochondrial d loop and the origin of modern

    humans. Nature, 378, 489.

    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, spe-

    cializing in aspects of African biodiversity and biogeography,examining species ranging from primates and elephants to

    tropical flora.