New Genetic Evidence on the Evolution of Chimpanzee …mkg62/assets/gonder,-disotell,-oates---2006...

International Journal of Primatology ( C© 2006)DOI: 10.1007/s10764-006-9063-y

New Genetic Evidence on the Evolutionof Chimpanzee Populations and Implicationsfor Taxonomy

Mary Katherine Gonder,1,2,4 Todd R. Disotell,3 and John F. Oates2

Received June 17, 2004; accepted July 1, 2005

Primatologists widely recognize chimpanzees as belonging to a single species,Pan troglodytes, which they traditionally have further divided into 3 sub-species: west African P. t. verus, central African P. t. troglodytes, and eastAfrican P. t. schweinfurthii. Previously, we suggested that the phylogeo-graphic history of chimpanzees may be different from that implied by thewidely used taxonomy of the species. We based the suggestion on only a lim-ited sample of haplotypes from the first hypervariable region (HVRI) of mi-tochondrial (mt)DNA from chimpanzees in Nigeria. We have now compileda more geographically comprehensive genetic database for chimpanzees, in-cluding samples obtained near the Niger and Sanaga Rivers. Our database iscomposed of 254 HVRI haplotypes from chimpanzees of known geographicorigin, including 79 unique HVRI haplotypes from chimpanzees living inNigeria and Cameroon. The genetic data provide clear evidence that a majorphylogeographic break between chimpanzee lineages occurs near the SanagaRiver in central Cameroon and suggest the need for a reclassification of chim-panzees.

KEY WORDS: chimpanzees; chimpanzee subspecies; mtDNA; phylogeography; populationstructure.

1Department of Biology, University of Maryland, College Park, Maryland.2Department of Anthropology, Hunter College and Graduate Center, City University of NewYork, 695 Park Avenue, New York, New York.

3Department of Anthropology, New York University, 100 Rufus D. Smith Hall, 25 WaverlyPlace, New York, New York.

4To whom correspondence should be addressed at Department of Biology, University ofMaryland, College Park, Maryland; e-mail: [email protected].

0164-0291/06 C© 2006 Springer Science+Business Media, Inc.

Gonder, Disotell, and Oates

INTRODUCTION

Several studies suggest that chimpanzees, whose genome is remark-ably similar to our own (Chen and Li, 2001; Wildman et al., 2003), possess2–6 times the genetic diversity of humans (Kaessmann et al., 1999; Kitanoet al., 2003; Stone et al., 2002). Studies of intraspecific variation among nat-ural populations of chimpanzees have lagged far behind those of humans,but they are important in clarifying differences and similarities in the evolu-tionary histories of contemporary populations of both species. Recently, thestudy of chimpanzee evolutionary biology has benefited enormously fromstudies using DNA from hairs shed into the sleeping nests of wild chim-panzees (Gagneux et al., 1999; Goldberg and Ruvolo, 1997; Morin et al.,1994). The studies have largely relied on analyses of haplotypes of the firsthypervariable region (HVRI) of mitochondrial DNA (mtDNA), which isa small maternally inherited locus that does not undergo recombination(Gray et al., 1999). The genetic studies have raised questions about intra-and intercommunity relationships among chimpanzees, and have examinedhow forest dynamics, geography, and demographic events have influencedtheir distribution and recent evolution.

For several decades, primatologists have widely recognized 3 sub-species of chimpanzees: Pan troglodytes troglodytes Blumenbach, 1799 fromcentral Africa, P. t. schweinfurthii Giglioli, 1872 from east Africa, and P.t. verus Schwarz, 1934 from west Africa (Fig. 1) (Hill, 1967, 1969). Thewidely accepted view of chimpanzee distribution patterns suggests that Pantroglodytes verus ranges from Senegal to western Nigeria. The DahomeyGap, a dry-forest zone covering present-day eastern Ghana, Togo, andBenin, divides Pan troglodytes verus into 2 populations. The lower NigerRiver, in Nigeria, separates Pan troglodytes verus and P. t. troglodytes. Pantroglodytes troglodytes ranges from eastern Nigeria to the Ubangi River inthe Democratic Republic of Congo (the former Zaire), and as far south asthe Congo River. The Ubangi River separates Pan troglodytes troglodytesand P. t. schweinfurthii. Pan troglodytes schweinfurthii ranges from theUbangi River and as far east as the western Rift Valley (Hill, 1967, 1969;Teleki, 1989).

Our previous analyses of all available HVRI haplotypes for wild chim-panzees have challenged the widely accepted taxonomy of the species andsuggest that chimpanzees in Nigeria and Cameroon may provide a key tounderstanding the recent evolutionary history of the species (Gonder et al.,1997). We based the suggestion on only a limited sample of haplotypesfrom chimpanzees in Nigeria. Therefore, we sequenced a 346-bp fragmentof HVRI from chimpanzees located near several putative biogeographicboundaries throughout Nigeria and Cameroon. We combined unique

Evolution of Chimpanzee Populations

Fig. 1. Widely accepted taxonomy of chimpanzees (Pan troglodytes) adapted fromHill (1967, 1969). Question marks (?) denote rivers that may separate chimpanzeesubspecies proposed with little evidence. Sampling locations of chimpanzee HVRIhaplotypes from previous studies are denoted by letters. We obtained HVRI hap-lotypes from previous studies from GenBank (Gagneux et al., 1999; Goldberg andRuvolo, 1997; Gonder et al., 1997; Morin et al., 1994).

haplotypes for analysis with HVRI haplotypes from other studies availablefrom GenBank. Geographic locations of samples from previous studies arein Fig. 1.

METHODS

Sample Collection

We obtained all genetic material from hairs shed in chimpanzee sleep-ing nests and from samples excised from preserved skins using standard pro-cedures (Gonder, 2000). Gonder collected 210 samples of hair from chim-panzee sleeping nests at 16 different locations across southern Nigeria andCameroon from a wide array of habitats (Fig. 1). Details about each sam-pling location are in Table I. We chose study locations to maximize sam-pling coverage across potential biogeographic barriers including west andeast of the Niger River, north and south of the Cross River, both sides of


Tab

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eria

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land

moi

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rest

rem

nant

(Agb

elus

i,19

94)

N07

◦ 22.

103′

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◦ 21.

869′

6/2

2O

wo

For

estR

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Low

land

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rest

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nant

(Agb

elus

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94)

Not

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03

&4

Cro

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Nat

iona

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PL

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and

mon

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fore

st(O

ates

etal

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90)

N06

◦ 23.

8′E

9◦19

.6′

6/6

5A

koh

Zan

toA

KZ

NL

owla

ndm

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slop

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com

m.)

N06

◦ 54.

644′

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◦ 53.

993′

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91)

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733′

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◦ 07.

515′

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626′

9/9


Tab

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m.)

N05

◦ 37.

281′

E11

◦ 40.

855′

10/1

0

14D

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-dun

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dm

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ove

fore

st(G

artl

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,197

2;P

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otte

tal.,

1996

)N

03◦ 3

5.82

0′E

09◦ 5

3.64

9′7/

7

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Wild

life

Res

erve

Cam

poH

eavi

ly-l

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dlo

wla

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st(M

itan

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tal.,

1996

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02◦ 1

9.37

7′E

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1′10

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1.40

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8.70

6′10

/7

a Map

loca

tion

ssh

own

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ig.2

.


Fig. 2. Sampling distribution of chimpanzee HVRIsequences in Nigeria and Cameroon. Note we collectedof shed hair samples from significantly decomposed nestsfrom map location 2, but we were unable to determineHVRI haplotypes for the nest hair collections.

the Cameroon Highlands, north and south of the Sanaga River, east of theMbaum River (Fig. 2, location 13), and into the Congo basin forest expansenear the Dja River (location 16). We searched each sampling location forevidence of chimpanzees, e.g., vocalizations, sightings, and evidence of re-cent chimpanzee activity, e.g., feeding sites, new trails, fresh or older nests.If we heard or saw chimpanzees, we tracked the individuals until dusk, leav-ing them in their nests. The following morning, we searched all accessiblefresh nests for shed hairs. If we could not establish evidence of recent chim-panzee activity in an area but found nests, we searched all accessible intactnests for shed hair. We collected 2–22 samples at each location.

DNA Extraction and Polymerase Chain Reaction

We isolated DNA from shed hair following well established pro-tocols (Gonder, 2000). We extracted DNA from single hairs wheneverpossible. If we could not obtain DNA suitable for cycle sequencingfrom single hairs, we used multiple hairs from single-nest collectionsto obtain suitable template for cycle sequencing. We used sequencesof pooled samples for analysis only when we obtained unambiguous


nucleotide sequencing results. We used 4 primers given in Goldbergand Ruvolo (1997) for polymerase chain reaction (PCR) and cycle se-quencing: L-16041, 5′-CTCTGTTCTTTCATGGGGAAGC-3′, L-16111,5′-ATTTCGTACATTACTGCCAGCC-3′; H16286, 5′-GGATGGATTTGACTGTAATGTGC-3′ and H-1411, 5′- TGTGCGGGATATTGATTTCA-3′. To obtain DNA template suitable for cycle sequencing, we amplifiedthe HVRI locus in 2 separate stages: an initial stage that amplifies theentire locus and a second amplification after post-PCR gel excision of thefirst PCR product. The second amplification either reamplified the entireregion or reamplified the region in 2 overlapping fragments, depending onthe quality of the initial PCR template.

We performed initial PCR reactions in 25-µl volumes, using 200–400 pg of genomic DNA extracted from shed hair, plucked hair, or pre-served skin. PCR reaction specifications for HVRI amplicons were: 10mM Tris-HCl, pH 8.3, 50 mM KCl, 2.0 mM MgCl2, 0.2 mM dNTPs,6.25 pmol of each primer, and 0.3125 U of Amplitaq R© DNA polymerase(Perkin-Elmer). We performed PCR reactions in a Perkin-Elmer thermo-cycler set to the following cycling parameters in thin-walled reaction tubes:94◦C for 45 s, 54◦C for 45 s, 72◦C for 1 min, for 35 cycles followed byan indefinite soak at 4◦C. We preheated the thermocycler to 94◦C be-fore loading to reduce nonspecific priming and extension during initialramping.

After initial amplification, we combined samples with 2 µl of 6 × load-ing dye and dried to 10 µl. We loaded the samples onto a 1% TRIS-Acetate-EDTA (TAE) low-melt agarose gel and electrophoresed for 45 min at 150V in TAE buffer. We lightly stained gels with ethidium bromide, rinsed inddH2O, and placed on a UV light table covered with plastic wrap. Usingsterile 1.5-ml disposable pipette tips, we removed small plugs from the cen-ter of the band region in each lane, and transferred them to 1.5-ml micro-centrifuge tubes containing 500 µl of ddH2O. We heated the tubes at 84◦Cfor 20 min to liberate the gel-bound HVRI fragment before reamplification.

We prepared 2 50-µl reactions for each sample using 2 µl of the dilutedinitial PCR product. PCR and thermocycling conditions were identical tothe initial amplification, except that we used 30 instead of 35 cycles dur-ing the PCR. After reamplification, we prepared all samples showing vis-ible single bands on an 8.0% polyacrylamide gel for DNA sequencing us-ing Qiaquick

TMPCR purification kits (Qiagen) to remove unincorporated

dNTPs and excess primers. Next, we assessed PCR product quantity andquality against known quantities of 100bp ladder (Promega Corp.) to deter-mine the suitabilty of all samples for DNA sequencing. Additional detailsabout PCR specifications are in Gonder (2000).


DNA Sequencing

We prepared PCR products for sequencing using QiaquickTM

PCRpurification kits (Qiagen) or by enzymatic digestion with shrimp alkalinephosphatase and exonulcease I (USB Corp.). We performed sequencingreactions using standard procedures with the 4 HVRI primers with ABI

TM

Big-DyeTM

terminator cycle-sequencing ready reaction kits. We analyzedall sequences using either an ABI

TM310 Genetic Analyzer, an ABI

TM3100

Genetic Analyzer, or an ABITM

377 DNA Sequencer (Applied Biosystems).We deposited all previously unpublished sequences in GenBank under theaccession numbers DQ140188-DQ140269.

Sequence Authentication

Nuclear mitochondrial pseudogenes (NUMTs) occur frequentlyamong hominoids (Collura and Stewart, 1995; Tourmen et al., 2002), andare particularly common among gorillas (Thalmann et al., 2004). Re-searchers have proposed several methods to authenticate mtDNA HVRIsequences, including comparing sequence product obtained from shortand long PCR fragments (>10 kb) for at least some samples (Thalmannet al., 2004). The largest NUMT in the human genome that contains a re-gion homologous to the HVRI is <10,000 bp in length (Tourmen et al.,2002). We authenticated the HVRI sequences by comparing sequenceproduct obtained from a 10,575-bp PCR product with sequence productobtained from the 346-bp locus for a panel of 20 chimpanzees from theCorriell Institute. We amplified the 10.6-kb fragment using Platinum R©Taq DNA Polymerase High Fidelity (Invitrogen Corp.) with the PCRprimer pair L10652, 5′-GCCATACTAGTCTTTGCCGC-3′and H4678, 5′-CAACCGCATCCATAATCCTT-3′. Names indicate the 5′ position of thefirst base pair of the primer sequence in the homologous region of the Cam-bridge Reference Sequence (Andrews et al., 1999). In all cases the HVRIsequence from both the large and small PCR products were identical (datanot shown), suggesting that NUMT contamination in the HVRI data is un-likely. Other factors make the likelihood of NUMT contamination small.We used species-specific PCR primers and sequencing primers, which pro-duce authentic HVRI sequences in chimpanzees (Thalmann et al., 2004).In addition, the ratio of nuclear to mtDNA is usually very low in DNA iso-lated from shed hair, which improves the chances of recovering an authenticHVRI sequence (Morin et al., 2001).


Sequence Analysis

We imported electropherograms to AutoAssembler R© (AppliedBiosystems) and Sequencher version 4.2 (Gene Codes Corp.). We alignedHVRI sequences by visual inspection of the assembled sequences. Weobtained chimpanzee HVRI sequences from other studies from Gen-Bank under the accession numbers L35381–L35443, U77186–U77293,AFO59042–AFO59052, and AF137481–AF137412. We obtained humanHVRI sequences from the Human Mitochondrial Genome Database(mtDB) at http://www.genpat.uu.se/mtDB/sequences.html. We alignedsequences from GenBank and mtDB with reference to sequences pro-duced for the study. Sequences we produced and used correspond to bases16,048–16,391 of the human mtDNA Cambridge Reference Sequence(Andrews et al., 1999).

Phylogenetic Analysis

We used NONA (version 2.0) (Goloboff, 1999) and PAUP∗ (version4.0b10) (Swofford, 2002) to construct a strict consensus of the 4000 mostparsimonious trees obtained using unordered and uniformly weighted char-acters in 100 replicate random addition heuristic searches via a tree bisec-tion and reconnection (TBR) branch swapping algorithm for all the HVRIhaplotype data. We saved the 20 shortest trees in each random additionreplicate and swapped the branches via TBR. We measured topological re-liability by performing 10,000 stepwise-addition bootstrap replicates withresampling.

We constructed a neighbor-joining tree using a distance matrix cor-rected via the Tamura and Nei model of nucleotide substitutions with γ dis-tributed rates (α= 0.6) in PAUP∗ (version 4.0b10) (Swofford, 2002; Tamuraand Nei, 1993). We used 10,000 jackknife replicates with 50% deletion ineach replicate to reconstruct the neighbor-joining tree with confidence lim-its. We determined the α value of 0.6 using the parsimony-based approxi-mations of γ parameters in PAUP∗.

We constructed maximum likelihood trees for the HVRI sequencedata via Fast DNAml (Felsenstein, 1981; Olsen et al., 1994) using empiri-cal base frequencies and 3:1, 6:1, and 10:1 transition:transversion (TS:TV)ratios. The overall tree topology did not change when we used differ-ent TS:TV ratios. We rooted all HVRI haplotype trees with 2 humanand 2 bonobo HVRI sequences. We traced character state changes alongthe branches of each tree via MacClade (version 4.06) (Maddison andMaddison, 2000).


TMRCA Analysis

To date the time to most recent common ancestor (TMRCA) for thechimpanzee HVRI, we estimated mutation rate (µ) as 4.7 × 10−8 substi-tutions/site/yr from the mean genetic distance between 1 chimpanzee and186 human (Ingman and Gyllensten, 2003) HVRI haplotypes (0.28 sub-stitutions/site). We assumed that humans and chimpanzees shared a lastcommon ancestor 6 million yr ago (Ma) based on paleontological evidencethat hominids were present in Chad by 6–7 Ma (Vignaud et al., 2002) andgenetic evidence of a divergence time between humans and chimpanzeesbetween 4.6 and 6.2 Ma (Chen and Li, 2001). We calculated TMRCAestimates for each putative chimpanzee lineage from mean genetic dis-tances inferred from a distance matrix corrected via the Tamura and Neimodel of nucleotide substitutions with γ distributed rates (Tamura andNei, 1993).

RESULTS

Phylogenetic Analysis

We obtained 79 unique HVRI haplotypes from chimpanzees in Nigeriaand Cameroon. We combined the HVRI haplotypes for phylogenetic anal-yses with 175 HVRI haplotypes from chimpanzees of known geographicorigin in Africa. The resulting alignment is 346 bp in length and contains199 polymorphic sites. The chimpanzee HVRI haplotypes differ on averageby 69 ± 30 substitutions (52 transitions and 17 transversions) uncorrectedfor multiple substitutions. For comparison, the Human MitochondrialGenome Database (mtDB) contained 186 human orthologous sequencesfrom diverse populations that differ on average by 9 substitutions (8 tran-sitions and 1 transversion) uncorrected for multiple substitutions. Inser-tion/deletion events occurred at 3 positions among the chimpanzee HVRIhaplotypes, but we did not use length polymorphisms for phylogeneticreconstruction.

Phylogenetic hypotheses for the HVRI haplotypes are in Figs. 3–5. Thetopologies of all the HVRI haplotype trees suggest that chimpanzees aredivided into 2 large monophyletic groups. Clade A is composed largely ofchimpanzees from Upper Guinea, Nigeria, and western Cameroon, whileclade B is almost entirely composed of chimpanzees from western equa-torial Africa and eastern Africa. The ranges of these lineages converge incentral Cameroon near the Sanaga River. HVRI haplotypes from samplescollected 10 km north of the Sanaga River (Fig. 2, map location, 12) and


those from 20 km south of the Sanaga River near the Douala-Edea For-est Reserve (map location, 14) belong to clade A and clade B, respectively.HVRI haplotypes from samples obtained at map location 13, 50 km east ofthe main tributary of the Sanaga, are equally divided between clade A andclade B. One HVRI haplotype from southern Cameroon (map location 14)is present in the eastern Nigeria and western Cameroon group. Detailedinformation about clades A and B, including the name and phylogeneticposition of each HVRI haplotype, is in Appendices 1–3.

The tree topologies in Figs. 3 and 4 suggest that within clade A, 2 mono-phyletic groups are present: 1 group composed of chimpanzees from UpperGuinea and western Nigeria (clade A1) and 1 group of chimpanzees fromeastern Nigeria and western Cameroon (clade A2). However, when we an-alyzed the data via a maximum likelihood criterion (Fig. 5), HVRI hap-lotypes of chimpanzees located in Upper Guinea formed a monophyleticgroup within the Nigeria and western Cameroon cluster.

HVRI haplotypes of chimpanzees in clade B do not consistently clusterinto 2 monophyletic groups that correspond closely with their geographicaldistribution (Fig. 3; Appendices I and III), suggesting that chimpanzees inwestern equatorial Africa and eastern Africa may share a relatively recentrelationship or have experienced high rates of migration. In addition, HVRIhaplotypes of 2 chimpanzees from southeastern Nigeria (map locations 3and 4) cluster within a western equatorial African chimpanzee group. TwoHVRI haplotypes from chimpanzees living in Cameroon consistently clus-ter with chimpanzees from eastern Africa.

We traced character state changes along the branches of each tree. Fivefixed substitutions (1 transition and 4 transversions) support the branchesdividing clade A from clade B. The low ratio of transitions to transversionsis probably due to saturation at mutational hotspots along the HVRI locus(Meyer et al., 1999). Several substitutions are nearly fixed in each group.Within clade A, the branch separating Upper Guinea chimpanzees (cladeA1) and those in eastern Nigeria and western Cameroon (clade A2) is sup-ported by 4 fixed transitions in the trees from Figs. 3 and 4. Several moresubstitutions are nearly fixed within each group. We detected no fixed nu-cleotide differences that divide HVRI haplotypes of chimpanzees in west-ern equatorial Africa from those in eastern Africa.

TMRCA Analysis

Table II contains our estimates of the time of the most recent com-mon ancestor (TMRCA) for each putative chimpanzee lineage calcu-lated from the corrected pairwise distance matrix used to construct the


HVRI haplotype tree in Fig. 4. The mean genetic distance betweenall the chimpanzee HVRI haplotypes (9.25 × 10−2) yields a TMRCA of991 ± 434 thousands of yr ago (Ka) for Pan troglodytes as a whole, assum-ing a rate of 4.7 × 10−8 substitutions/site/yr. Within the chimpanzee lin-eage, those residing in western equatorial Africa (clade B1) shared themost ancient mitochondrial ancestor, estimated at 960 ± 403 Ka. Chim-panzees in eastern Nigeria and western Cameroon (clade A2) may haveshared a mitochondrial ancestor 681 ± 310 Ka; populations in UpperGuinea and western Nigeria (clade A1) may have shared a mitochon-drial ancestor 558 ± 248 Ka; and populations in eastern Africa (clade B2)may have shared the most recent mitochondrial ancestor, at 279 ± 124Ka.

DISCUSSION

Our results strongly suggest that 2 deeply divergent lineages of chim-panzees are present in African forests, 1 in central and eastern Africa and1 in western Africa. The 2 lineages diverged ≥ 500 Ka but their present ge-ographic ranges converge in central Cameroon. It appears that the SanagaRiver, or some historical environmental discontinuity in the vicinity of theSanaga, has played an important role in limiting gene flow between them.Additional population genetic analyses of the HVRI haplotypes furthersuggest that the Sanaga River plays an important role in limiting gene flowbetween chimpanzees in Nigeria and western Cameroon and those livingin Cameroon south and east of the Sanaga River. For example, the mi-gration rate calculated from HVRI haplotypes of chimpanzees living oneither side of the Sanaga River is lower than among any other popula-tions of chimpanzees in close proximity to one another. Chimpanzees oneither side of the Sanaga exchange 1.1 migrants/generation (Gonder, 2000).In contrast, chimpanzees in eastern Africa exchange an average of 25.8migrants/generation between different regional forests in eastern Africaand 3.38 migrants/generation between sampling locations (Goldberg and

←−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−Fig. 3. HVRI parsimony tree for chimpanzees at the HVRI locus constructed via NONA(Goloboff, 1999). The tree represents a strict consensus of 4000 trees examined in a 100 repli-cate random addition heuristic search using TBR branch swapping. We determined topolog-ical reliability by 10,000 bootstrap replicates with resampling. Crosses ( + ) indicate samplesobtained from outside the main geographic distribution of most members of that group. As-terisks (∗) show the location of samples obtained at map location 13 from Fig. 2. Clades C andD are bonobo and human outgroups, respectively. Name and phylogenetic position of eachchimpanzee HVRI haplotype are in Appendix I.


Table II. Time since most recent common ancestor (TMRCA) among humans andchimpanzees

HVRI haplotypes n Substitutionsa % of human estimateb TMRCAKYAc

Homo sapiens 186 10 ± 5 310 ± 155Pan troglodytes 254 32 ± 14 320 991 ± 434Clade A: Upper

Guinea + easternNigeria and westernCameroon

87 30 ± 13 300 929 ± 403

Clade A1 38 18 ± 8 180 558 ± 248Clade A2 49 22 ± 10 220 681 ± 310

Clade B: westernEquatorial + eastern

167 18 ± 8 180 557 ± 248

Clade B1 39 31 ± 13 310 960 ± 403Clade B2 128 9 ± 4 90 279 ± 124

aAverage substitution rate inferred from the distance matrix used to construct theneighbor-joining tree in Fig. 4.bCalculated by dividing the average number of substitutions by the number of substi-tutions calculated from the human HVRI sequences using a distance matrix constructedusing a Tamura and Nei (1993) distance correction with gamma distributed rates. We cal-culated the average number of substitutions among humans and TMRCA from a globalsample of 186 HVRI haplotypes (Ingman and Gyllensten, 2003).cWe calculated all divergence dates for chimpanzees, expressed in thousands of years ago(kya), assuming a mutation rate (µ) of 4.7 × 10−8.

Ruvolo, 1997). Complementary analyses of microsatellite variation alsosupport the proposition that the Sanaga is an important barrier to geneflow between chimpanzee populations in western and southern Cameroon(Gonder and Disotell, 2006).

Our sample sizes for each sampling location were necessarily small(<11), owing to the difficulties of collecting and using DNA from noninva-sive sources such as shed chimpanzee hair. Thus we may have missed someof the genetic variation in each lineage, particularly rare haplotypes that fallinto unexpected clades like those that occur east of the Mbaum River thatcluster into clade A, BYM3, CRNP 6, CRNP 12, and DBR7. The cluster-ing pattern of these samples with samples from distant geographic locationssuggests that the historical relationships between chimpanzees in Nigeria

←−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−Fig. 4. 10,0000 replicate jackknife neighbor-joining tree of the chimpanzee HVRI locus. Weconstructed the tree using Tamura and Nei’s distance correction in PAUP∗ (Swofford, 2002;Tamura and Nei, 1993). Confidence limits for all branches in the neighbor-joining tree rangedfrom 85% to 100%. Crosses ( + ) indicate samples obtained from outside the main geographicdistribution of most members of that group. Asterisks (∗) show the location of samples ob-tained at map location 13 from Fig. 2. Clades C and D are bonobo and human outgroups,respectively. Name and phylogenetic position of each chimpanzee HVRI haplotype are inAppendix II.


Fig. 5. Simplified maximum-likelihood tree of chim-panzee HVRI haplotypes. The tree represents the mostlikely topology of 218,608 tree topologies examined (lnlikelihood = − 5459.55) via Fast DNAml (Felsenstein,1981; Olsen et al., 1994). Clade A shows the relation-ship between chimpanzee HVRI haplotypes from UpperGuinea, eastern Nigeria, and western Cameroon. Clade Bis composed of chimpanzee HVRI haplotypes from west-ern equatorial and eastern Africa. Crosses ( + ) indicatesamples from outside the main geographic distribution ofmost members of that group. Asterisks (∗) show the lo-cation of samples obtained at map location 13 from Fig.2. Clades C and D are bonobo and human outgroups, re-spectively. Name and phylogenetic position of each chim-panzee HVRI haplotype are in Appendix III.


and Cameroon may be more complex than just being separated on eitherside of the Sanaga River. They belong to the same species, so it is not en-tirely surprising to find some unexpected overlap of HVRI haplotypes in thevicinity of the Sanaga. Little is known about the history of the Sanaga Riverbut researchers have proposed it has a considerable impact on the distribu-tion of many other animals. Among the primates the Sanaga River may bean important boundary between Arctocebus calabarensis/A. aureus, Euoti-cus pallidus/E. elegantulus, Cercopithecus erythrotis/C. cephus, C. nictitansmartini/C. n. nictitans, C. pogonias pogonias/C. p. grayi, and Mandrillus leu-cophaeus/M. sphinx; it also limits the distribution of Colobus satanas (Gart-lan and Struhsaker, 1972; Groves, 1993; Grubb, 1982, 1990; Kingdon, 1989;Oates, 1988).

Relationships among chimpanzees within the western African andcentral-eastern African lineages are more complex. Chimpanzee popula-tions in western equatorial Africa carry mtDNA haplotypes that are morediverse and more ancient than in any other population. The observationis consistent with the findings of a survey of genetic diversity at 9 auto-somal intergenic regions of chimpanzees in western Africa compared tothose in western equatorial Africa, which suggests that chimpanzees fromwestern equatorial Africa have higher nucleotide diversity and a larger ef-fective population size than chimpanzees in western Africa (Fischer et al.,2004). The branching patterns in the HVRI haplotype trees also suggestthat chimpanzee populations in western equatorial Africa share a relativelyrecent genetic relationship with chimpanzees living in eastern Africa, and2 HVRI haplotypes of chimpanzees from southern Cameroon are nearlyidentical to HVRI haplotypes of chimpanzees from eastern Africa. Re-searchers have reported similar observations in morphological surveys.Comparisons of crania and mandibles among chimpanzees from westernequatorial Africa and eastern Africa suggest that the 2 groups share con-siderably more overlap at landmark features relative to chimpanzees fromUpper Guinea (Braga, 1998; Shea and Coolidge, 1988; Taylor and Groves,2003). Therefore, chimpanzees in western equatorial Africa and easternAfrica may share a closer relationship to each other than either groupdoes to chimpanzees from Upper Guinea. Differences among east Africanchimpanzees remain unresolved. In contrast, an analysis of craniometricmarkers suggests that they may be divided into 2 distinct groups: a north-western group (from Oubangui, Uele, and Ituri districts) and a southeast-ern group (from Uganda, Maniema, and Marungu), both of which arewell differentiated from chimpanzees in central/western equatorial Africa(Groves, 2005).

Our phylogenetic inferences consistently divide west African chim-panzees into 2 separate groups, 1 in the westernmost Upper Guinea forests,


and 1 further east in Nigeria and western Cameroon, suggesting that the 2populations in west Africa historically have been more isolated from eachother than chimpanzees in western equatorial African forests have beenfrom those in eastern Africa. At this point we are unable to say where themain geographic divide occurs between the 2 groups of west African chim-panzees. Chimpanzees are rare in western Nigeria, and our sample of chim-panzees from that area is small. Scientists have not sampled chimpanzeesin western Ghana. More data are needed before we can state more con-fidently whether a phylogeographic division exists between west Africanchimpanzees at the Niger River, or at the Dahomey Gap in the vicinity ofTogo, or whether chimpanzees in western Nigeria form a distinct geneticpopulation. Groves (2001) reported that skulls from western and easternNigeria were alike, but his sample size was too limited to reach a definitiveconclusion as to whether chimpanzees on either side of the Niger River aresignificantly different from each other.

Our findings suggest 2 taxonomic alternatives. HVRI haplotype datamost strongly support dividing chimpanzees into just 2 subspecies: Pantroglodytes vellerosus in western Africa and P. t. troglodytes in central andeastern Africa. The name vellerosus, which Gray gave in 1862 to a specimencollected in Cameroon (Gray, 1862), takes taxonomic precedence oververus, named 72 yr later. The ranges of these 2 forms are in Fig. 6a. Alter-natively, if the division of chimpanzees within west Africa is regarded assufficient to be represented taxonomically, the 2 forms are Pan troglodytesverus in Upper Guinea and P. t. vellerosus in Nigeria and westernCameroon (Fig. 6b). Recognition of 2 subspecies in west Africa is appro-priate if the current taxonomy of western equatorial African and easternAfrican chimpanzees (Pan troglodytes troglodytes and P. t. schweinfurthii) ismaintained.

The taxonomy and distribution pattern in Fig. 6b has gained grow-ing support. Several authors have provisionally recognized Pan troglodytesvellerosus as a valid chimpanzee subspecies (Groves, 2001; Grubb et al.,2003; Kormos et al. 2003). However, Groves (2001) reported that whilechimpanzees from Nigeria appear to be different from other chimpanzees incertain cranial nonmetrical features, Nigerian chimpanzees were more simi-lar to chimpanzees from western equatorial Africa and eastern Africa com-pared to those from Upper Guinea. Increased sampling coverage in westernGhana and western Nigeria, analyses of additional genetic loci, and furthermorphological analysis are necessary to understand the phylogeographichistory of chimpanzees more fully.


Fig. 6. Two chimpanzee taxonomies inferred from the HVRI haplotype data.

ACKNOWLEDGMENTS

We thank the federal governments of Cameroon and Nigeria; thestate governments of Cross River, Ondo, and Ekiti in Nigeria; the NigerianConservation Foundation, the Pandrillus Foundation; Pronatura Interna-tional (Nigeria); the Wildlife Conservation Society; and the World WideFund for Nature for their support during sample collection in Cameroonand Nigeria by M. K. Gonder. We also thank 3 anonymous reviewersfor their insightful comments on the manuscript. The L.S.B. LeakeyFoundation, Primate Conservation, Inc., the National Science Foundation(Dissertation Improvement Award, Graduate Research Fellowship, andNYCEP Research Training Grant), and the Wenner-Gren Foundationsupported the research. We exported all samples from Africa under CITES


exportation permits (Cameroon: 0172/PE/MINEF/DFAP/SL/SLP; Nigeria:FEPA/LSN/68/T/39) and imported them under U.S. CITES (US810330)and USDA (97-418-2) importation permits.

APPENDIX 1. Clades A and B of the parsimony tree shown in Fig. 3. Crosses (+) denote HVRI haplotypes ofsamples collected outside the geographic area given to right of the tree. Astericks (*) denote HVRI haplotypes of samplesfrom Man’bra (Fig. 2, map location 13.).


APPENDIX II. Clades A and B of the neighbor-joining tree shown in Fig. 4. Crosses (+) denote HVRI haplotypes ofsamples collected outside the geographic area given to the right of the tree. Astericks (*) denote HVRI haplotypes of samplesfrom Man’bra (Fig. 2, map location 13.).


APPENDIX III. Clades A and B of the maximum-likelihood tree shown in Fig. 5. Crosses (+) denote HVRI haplo-types of samples collected outside the geographic area given to the right of the tree. Astericks (*) denote HVRI haplotypesof samples from Man’bra (Fig. 2, map location 13.).


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