American Journal of Primatology 64:261–275 (2004)

RESEARCH ARTICLE
Mitochondrial DNA Genealogy of Chimpanzees in the
Nimba Mountains and Bossou, West Africa
MAKOTO K. SHIMADA1, SACHIKO HAYAKAWA2, TATYANA HUMLE3, SHIHO FUJITA4,SATOSHI HIRATA5, YUKIMARU SUGIYAMA6, and NARUYA SAITOU1n1
Division of Population Genetics, National Institute of Genetics, Mishima, Japan
2
Section of Ecology, Primate Research Institute, Kyoto University, Inuyama, Japan3
Department of Psychology, University of Stirling, Stirling, United Kingdom
4
Division of Veterinary Medicine, Faculty of Agriculture, Gifu University, Gifu, Japan
5
Great Ape Research Institute, Hayashibara Biochemical Laboratories Inc., Tamano,Japan
6
Faculty of Humanities, Tokai-Gakuen University, Nagoya, JapanThe chimpanzee populations of the Bossou and Nimba regions in West
Africa were genetically surveyed to 1) reveal the genetic relationshipbetween the Bossou and Nimba populations, and 2) elucidate theevolutionary relationship between the Bossou-Nimba and other West
African populations. The chimpanzee group at Bossou is characterized byits small population size, no evidence of contact with neighboring
populations, and no female immigration. It is believed that most females
and adolescent males emigrate from this population. To reveal the genetic
signature of these characteristics, we examined the genetic diversity of
Bossou and two neighboring populations (Seringbara and Yealé) in the
Nimba Mountains by sequencing approximately 605 bp of the mitochondrial DNA (mtDNA) control region. A total of 20 distinct mtDNA variants
were observed from 56 sequences of noninvasively collected, anonymous
samples. Nucleotide diversity in the Nimba Mountain populations was
0.03–0.04, and did not differ significantly from that in the Bossou
population. Very few mitochondrial variants are shared among the sites
sampled, which suggests that there is little gene flow involving mtDNA.
Nevertheless, no clear population structures were revealed in either
population. A comparison with published sequences from West African
Contract grant sponsor: Ministry of Education, Science, Sports, and Culture, Japan; Contract grant
numbers: 14011248; 10041168; 10CE2005; Contract grant sponsor: National Institute of Genetics
COE; Contract grant sponsor: Nissan Science Foundation; Contract grant sponsor: Inamori
Foundation.

Makoto K. Shimada’s present address is Department of Genetics, Rutgers University, Piscataway,
New Jersey.
Tatyana Humle’s present address is Department of Psychology, University of Wisconsin, Madison, WI.
nCorrespondence to: Naruya Saitou, Division of Population Genetics, National Institute of Genetics,
1111 Yata, Mishima, 411-8540, Japan. E-mail: nsaitou@genes.nig.ac.jp
Received 9 November 2003; revised 13 June 2004; revision accepted 18 June 2004
DOI 10.1002/ajp.20077
Published online in Wiley InterScience (www.interscience.wiley.com).

r 2004 Wiley-Liss, Inc.

262 / Shimada et al.

chimpanzees (Pan troglodytes verus) indicates that the variants observed in the Bossou and Nimba regions are scattered throughout thesubspecies, rather than clustered according to geographic region. Thissuggests that the Bossou-Nimba populations derived only recently fromthe common ancestral population of the West African chimpanzees, anddid not pass through a bottleneck. Am. J. Primatol. 64:261–275, 2004.
r 2004 Wiley-Liss, Inc.

Key words: chimpanzee; Bossou; Nimba; mtDNA; genetic diversity;
noninvasive sampling

INTRODUCTION
The wild population of Pan troglodytes verus at Bossou, Republic of Guinea,
is the smallest wild chimpanzee community under long-term study. It has
consisted of about 20 individuals since the start of observations in 1976
[Sugiyama, 1999]. Chimpanzees in Bossou mostly confine their activity to a corearea of about 6 km2. Since the core area is surrounded by savanna and a little
gallery forest, the Bossou chimpanzee group has rarely been observed toencounter neighboring groups. The Nimba Mountains area, located on theborder between the Republic of Guinea, Ivory Coast, and Liberia, is separated bysavanna vegetation from Bossou, the closest neighboring population (Fig. 1).
UNESCO designated 220 km2 of the Nimba Mountains regions as a ‘‘strict nature
reserve,’’ and the chimpanzee population is considered to be protected in thisreserve [Matsuzawa & Yamakoshi, 1996].
Contrary to the commonly recorded norms of chimpanzee behavior, female
immigration has not been observed in the Bossou group. However, both
sexes probably emigrate [Sugiyama, 1999]. This migration pattern is clearly
different from other groups under long-term study [Gagneux et al., 2001; Goodall,
1986; Nishida et al., 1990]. Sugiyama [1999] investigated the relationshipbetween this unusual migration pattern and the habitat isolation at Bossou
from a behavioral perspective. He hypothesized that the absence of neighboring competitors negates the need for the alpha male to form alliances withother within-group males for support. Therefore, the other group males take on
the role of competitors for limited food resources and females. Sugiyama [1999]
also suggested an alternative hypothesis that the observed difference insocial structure and migration patterns may be a characteristic of this subspecies. Conclusive answers await further analysis; however, if human land use
continues to encroach on the chimpanzee habitat, this valuable research
population may be lost. Our investigation of the genetic relationship of the
Bossou group with surrounding groups in the Nimba Mountains may provide
useful data for a conservation plan for the chimpanzee populations in thisarea. The Bossou group data will be compared with results from several Nimba
Mountain populations that have been studied already [Matsuzawa et al., 1999],
including those at Goera, Yealé, and Seringbara [Humle & Matsuzawa, 2001;
Matsuzawa & Yamakoshi, 1996; Shimada, 2000; Sugiyama, 1995]. Within thisframework, we investigate the genetic relationship between the Bossou groupand other Nimba Mountain populations, and examine the matrilineal andgeographical similarities among these populations to understand the long-term
migration pattern of the Bossou-Nimba region as compared to the entire
Pan troglodytes verus subspecies.

mtDNA of Chimpanzees in Nimba and Bossou / 263

Fig. 1. Map of sampling sites. This map is based on UNESCO [1998].

MATERIALS AND METHODS

Sample Collection
Noninvasive samples, including hair, feces, and wadges (chewed fruit
remnants), were collected from wild chimpanzees living at Seringbara, Yealé,and Goera in the Nimba Mountains, and neighboring Bossou (Fig. 1). We collectedhairs from night beds by climbing the tree to the bed and taking hairs by forceps
after the chimpanzee left. (We use the term ‘‘bed’’ because in the ecologicalliterature, ‘‘nest’’ refers to a shelter for bearing and nursing offspring.) We stored
hairs collected from one bed in a plastic disposable tube, after adding absoluteethanol to the tube. We collected the feces by wiping the surface of the feces with acotton swab soaked in a saline/EDTA (1 mM) solution, and washing the cottonswab in 2 ml of the saline/EDTA solution. We then added 10 ml of absoluteethanol. We collected the wadges using forceps, and stored them in a plastic
disposable tube after adding absolute ethanol. These samples were stored at room264 / Shimada et al.

temperature, avoiding sunlight in the field. Hair samples were transferred to a
201C freezer, and feces and wadge samples were stored in a refrigerator in
the laboratory. While the chimpanzees in the Nimba Mountains have not been
identified individually, all of the samples from individuals in the Bossou
population were from known and identifiable individuals [Sugiyama, 1999]. At
the Seringbara site, local guides from Bossou village collected samples under the
direction of M.K.S. during October–November 1999, except for eight samples thatwere collected by S.F. and S.Hi during January–February 2000. At the Yealé site,
local guides collected samples under the direction of T.H. during February–
December 1999. At the Goera site, M.K.S. collected samples during February–
March 1999 and October 1999. Anonymous sampling was also conducted in
Bossou during January–March 1999 by S.Ha and M.K.S. to compare the resultswith sampling from known individuals.

DNA Extraction
We used different methods of DNA extraction according to the sample type(hair, feces, and wadges), as follows:

Hair.
We extracted DNA from single hairs using Isohair (Nippon Gene Co., Ltd.,Tokyo, Japan) according to the manufacturer’s instructions. The hair root was
visible in most samples.
Feces.
DNA was extracted with the use of the QIAmps DNA stool kit (Qiagen,Hilden, Germany) according to the manufacturer’s instructions, with thefollowing modifications [Morin et al., 2001] (Shimada et al., unpublished results):
after centrifugation (800  g, 10 min at room temperature), the precipitate wassuspended in 1.6 ml ASL buffer, and incubated at room temperature for 30–60
min. DNA was eluted in AE buffer for 20–30 min.
Wadges.
We followed the method of Sugiyama et al. [1993], with a slight modification.
Instead of using 95% ethanol and EDTA, we used absolute ethanol to store thesamples.
Mitochondrial DNA Sequence Determination
Shimada et al. (unpublished results) characterized about 605 bp of thehypervariable region I of the mtDNA control region of 14 individuals in the
Bossou population. The primer sets used were L15926 (50 -TACACTGGTCTTGTAAACC-30 corresponding to positions 15326–15344 of the complete chimpanzee
mtDNA sequence of Horai et al. [1995], DDBJ/EMBL/GenBank accession number
D38113), and H16555 (50 -TGATCCATCGTGATGTCTTA-30 ; corresponding topositions 15971–15990 of D38113), and inner primer set; L15933 (50 GGTCTTGTAAACCGGAAACG-30 ; 15332–15351 of D38113) and H16538; 50 TCTTATTTAAGGGGAACGTGTG-30 , 15954–15975 of D38113).
The purified PCR products were sequenced directly from both ends with theuse of the ABI PRISMt BigDye terminator cycle sequencing kit (Perkin Elmer,
Wellesley, MA), and the same primers were used for PCR. Sequences weremtDNA of Chimpanzees in Nimba and Bossou / 265

generated with automated sequencers (ABI 377 or ABI310; Perkin Elmer,
Wellesley, MA).

Nucleotide Sequence Analyses
Published mtDNA sequences of wild chimpanzees were retrieved from the
DDBJ/EMBL/GenBank International Nucleotide Sequence Database through the
DDBJ website (www.ddbj.nig.ac.jp). We also obtained geographic information
regarding the published Pan troglodytes verus sequences by contacting authors.
Multiple alignment of nucleotide sequences was performed with the use of
CLUSTAL W [Thompson et al., 1994].
We constructed a phylogenetic network using the reduced median network
method [Bandelt et al., 1995] with NETWORK 3.0.1.2 software (URL http://
www.fluxus-engineering.com). The neighbor-joining method [Saitou & Nei, 1987]
was used to construct a phylogenetic tree.
Since the sample size of the Goera site was small, we did not include thispopulation in the population structure analysis. Therefore, only Bossou,
Seringbara, and Yealé were included in the analysis. Nucleotide diversity within
population, net number of nucleotide difference (DA), pairwise FST, and analysis
of molecular variance (AMOVA) [Excoffier et al., 1992] analyses were conductedwith the help of ARLEQUIN version 2.000 [Schneider et al., 2000]. For the
calculation of DA and FST, the following formulae were used, respectively:
DA ¼ ^ 12  ð^ 1 þ ^ 2 Þ=2;
where ^ 1 and ^ 2 are estimates of the average number of nucleotide substitutions
for a randomly chosen pair of variants in populations 1 and 2, respectively, and^ 12 is the estimate of the average number of nucleotide substitutions between
variants from populations 1 and 2 [Nei & Li, 1979]:
FST ¼ ðf0  f1 Þ=ð1  f1 Þ;
where f0 is the probability of identity by descent of two different genes drawn
from the same population, and f1 is the probability of identity by descent of two
genes drawn from two different populations [Slatkin, 1991].
To test the significance of the variance component and F-statistics, 1,000
random permutations were generated and compared with observed values.
RESULTS

Success Rate of Nucleotide Sequence Determination
We collected three types of samples (hair, feces, and wadge) from four sites(Seringbara, Yealé, Goera, and Bossou). When we had easy access to chimpanzeenight beds, we collected hair samples from used beds. Feces were not easy to find,except at the Bossou site, where we were able to distinguish chimpanzee
individuals and follow them to collect fresh feces. It was generally difficult tocollect samples at the Goera site because, as suggested by the field survey, thechimpanzee group did not use this site continuously [Shimada, 2000]. The
numbers of samples collected from Seringbara, Yealé, Goera, and Bossou were 26,
23, four, and 21, respectively. A total of 74 samples (58 hairs, four feces, and
12 wadges) were used.
Table I shows the success rates of DNA sequence determination for samples
collected at four sites, and for three sample types. The average success rate was
76%, though it varied depending on the sample type. The success rate was thehighest for hair samples (88%), followed by feces (50%) and wadges (25%). The

266 / Shimada et al.

low success rates for feces and wadges may be attributed to the condition of thesamples, which were probably highly degraded. In contrast, the hair samples werecollected from fresh night beds, so they probably had better-quality DNA.

Observed mtDNA Variants
We were able to determine ca. 605-bp nucleotide sequences of the
mtDNA control region from a total of 56 samples. Twenty distinct variants wereidentified from those sequences. The nucleotide sequence data were depositedin the DDBJ/EMBL/GenBank International Nucleotide Sequence Database underaccession numbers AB189236–AB189251. Table II shows the distribution of those
TABLE I. Success Rate of Sequence Determinationn

Sample type
Population

Hair

Feces

Wadge

Total

Seringbara
Yealé
Goera
BossouTotal
Rate (%)

23/25
7/11
1/1
20/21
51/58
88

–
2/2
0/2
–
2/4
50

0/1
2/10
1/1
–
3/12
25

23/26
11/23
2/4
20/21
56/74
76

n
Values to the left of the slash are numbers of samples from which sequences were successfully determined, andthose to the right of the slash are total number of samples used. Hyphen indicates no sample.

TABLE II. Variant Distribution Among the Four Locations

Sampling location
Variant

Seringbara

Yealé

GoeraBossou

S-1
S-2
S-3
S-4
S-5
S-6
S-7
Y-1
Y-2
Y-3
Y-4
Y-5
Y-6
Y-7
Y-8
Y-9
Bs-TJFKa
Bs-Na
Bs-Pa
Bs-Va
Bs-Ya
Total

8
5
2
1
3
1
3
0
0
0
0
0
0
0
0
0
0
0
0
0
0
23

0
1
0
0
0
0
0
1
1
2
1
1
1
1
1
1
0
0
0
0
0
11

0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
2

a

These variants will be reported elsewhere.

Total
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
10
1
4
5
0
20

8
6
2
2
3
1
3
1
1
2
1
1
1
1
1
1
11
1
4
5
0
56

mtDNA of Chimpanzees in Nimba and Bossou / 267

20 variants among the four sampling locations. Because most of thevariants were found at only one site, we named the variants after sampledsites. Variants S-1 to S-7 were mostly from Seringbara, while variants Y-1 toY-9 were exclusively found in Yealé. Although five distinct variants were foundin Bossou in our previous study (Shimada et al., unpublished results), we
did not find variant Bs-Y in this study. Three of the remaining four variants
were found exclusively at Bossou. The fourth, Bs-TJFK, was found at both Bossou
and Goera.
Table III shows the alignment of the 21 mtDNA variants from the Nimba
Mountains and Bossou. Only variant sites are presented. The variant names are
the same as shown in Table II. Figure 2 shows a phylogenetic network based on
the sequence data shown in Table III. We identified six major mitochondrial
lineages out of 21 variants, all but one of which are known in the Bossou
population (Shimada et al., unpublished results). The only one not found in the
Bossou population is the Y-3 variant. There are several reticulations in this
phylogenetic network. In particular, interior reticulations form two complexes:
one that connects clusters 1, 5, and 6 (RC156), and one that connects clusters 2, 3,
and 4 (RC234). Single site differences make both of the reticulation complexes
three-dimensional (16,233 bp and 16,323 bp for RC156, and 16,233 bp and 16,399
bp for RC234). Since recombination cannot occur in mtDNA, all of thereticulations were caused by parallel substitutions.

Nucleotide Diversity Among the Three Populations
Because the number of individuals from which the samples were collected
is unknown, we estimated nucleotide diversity within each population byconsidering two extreme possibilities: 1) minimum estimates were modeled
as if each sample collected came from a unique individual, and 2) maximum
estimates were constructed under the assumption that each individual bears aunique mitochondrial variant. Table IV shows these estimates, as well as theactual values for the Bossou population, in which samples were collected fromknown individuals (Shimada et al., unpublished results). The minimum estimate(0.034) was not far from the true value (0.038) for the Bossou population(insignificant in z statistics: z=0.138. As for the Seringbara and Yealé
populations, the differences between the minimum and maximum estimates
were insignificant z=0.242 and 0.006, respectively. The true value of thenucleotide diversity must lie between the minimum and maximum estimates.
The estimated values for the Seringbara and Yealé populations are between
0.029 and 0.043. Those values are not significantly different from the true value(0.038) for the Bossou population, and both maximum and minimum estimates
are not significantly different from the true value (0.038) for the Bossou
population (minimum estimate of Seringbara vs. Bossou; z=0.366, maximum
estimate of Seringbara vs. Bossou; z=0.102, minimum estimate of Yealé vs.
Bossou; z=0.190 d.f.=7.6, maximum estimate of Yealé vs. Bossou; z=0.182.
Therefore, the three chimpanzee populations are considered to have the same
nucleotide diversities.
Goldberg [1998] characterized mtDNA HV I region sequences (ca. 370 bp)
from anonymous hair samples collected from 19 Eastern chimpanzee(P. troglodytes schweinfurthi) populations, and calculated nucleotide diversity(minimum estimates) within each population. The nucleotide diversities of these
19 populations ranged from 0.009 to 0.030, with an average of 0.021. Thisestimate is somewhat lower than our estimate (0.03–0.04) for the Nimba and

S-1
S-2
S-3
S-4
S-5
S-6
S-7
Y-1
Y-2
Y-3
Y-4
Y-5
Y-6
Y-7
Y-8
Y-9
Bs-TJFK
Bs-P
Bs-V
Bs-Y
Bs-N

atacctgcacctgcc-ccttatccccttaactctcgcatccc-aagtctccctcactcatcctacacgttggcgtc...t..a....c..t-.t........cc.g..t.ta.....tc..a.................g...a..aat...
.ctt.cat.....ttct..cg.....ccggt..c...g..t.cg....ct..c.c.c.c......gt...aa..a.
...t.....t..a..-................t.........c.............c...................
...............-..........................-........t........................
...t.....t..a..-................tc........c.g...........c...................
.ctt.cat.....ttct..cg....tccggt..c...g..t.cg....ct..c.c.c.c......gt...aa..a.
............a..-.t......t.................c........t........................
.ctt.ca......ttct..cg.t...ccggt..c...g.tt.cg...tct..c.c.ctc.....t.t...aa..at...t.......cat.-...........c.g.ctc....c...c.g....tt......t.c..cg...ac.a.t...
.ctt.ca......ttct..cg....tccggt..c...g..t.cg....ct..c.c.c.c......gt...aa..a.
...............-...........c..............-............t....................
...t.....t..a..-...............ct.........c.............c...................
...t........a..-................t.........c.............c...................
tcttt.a.c...attct.cc....t..cg....g...g....-g.....t..ctt.ctcc-....gt..caa.aa.
.ctt.ca......ttct..cg....tccggt..c...g..t.cg....ct..c.c.c.c......gt...aa..at...t.c...t..a..-................t.........c...c.........c...................
...tt.a....c..t-.t........cc.g..t.ta.....tc..a.................g...a..aat...
.ctt..a...t.a.tc.t.c.c.tt.ccgg...c.atg....cg....ct....c.c.c..t...gt...aa..a.
.ctt.cat.....ttct..cg....tccggt..c...g..t.cgg...ct..c.c.c.c......gt...aa..a.
.cttt.a.....attct.cc....t..cg....c...g....-g.....t..ctt.ctcc.....gt..caa.aa.

Nucleotide position corresponding to Anderson Sequence (J01415)________________________________________________________________________________
1111111111111111111111111111111111111111111111111111111111111111111111111111
5555666666666666666666666666666666666666666666666666666666666666666666666666
9999111111111111111111112222222222222222222333333333333333333333334444445555
3377122334445567777788990222334466777889999001112224555566777888990346891133
Name
6902149470386760138927137235038901348480236340170378255702459017590857772658_______________________________________________________________________________________TABLE III. Nucleotide Sequences of mtDNA Variants Found in This Study

268 / Shimada et al.

mtDNA of Chimpanzees in Nimba and Bossou / 269
Bs-V

Cluster 4

Cluster3
S-7 Bs-Y
S-3

Y-9
Y-4

Y-2

Bs-N
Y-8
Cluster 2

Cluster 5
Bs-P

S-2

Cluster 6
Y-3

S-6
Bs-TJFK

Cluster 1

Y-7
S-4
Y-6
Y-5
S-1
S-5
Y-1

: Distance of one site difference

Fig. 2. A phylogenetic network of mtDNA hypervariable region I variants observed in chimpanzees
at the Bossou and Nimba regions. The root is located using bonobo (Pan paniscus) sequence as an
outgroup.

TABLE IV. Comparison of Three Types of Estimates of Nucleotide Diversity

Estimation method
Population

True

Minimuma

Maximumb

BossouSeringbara
Yealé

0.038
N.D.c
N.D.c

0.034
0.029
0.043

0.054
0.036
0.043

a

Calculated under the assumption that all samples come from different individuals.
Calculated under the assumption that each individual bears a different variant.
c
Not determined because individual identification was not possible.
b

Bossou populations. However, since the sequence regions analyzed in these twostudies differed, a simple comparison may not be meaningful. Gagneux et al.
[1999] also reported nucleotide diversity (minimum estimates) based on a total of270 / Shimada et al.

292 sequences. They found that the Eastern chimpanzees had lower diversity(range ¼ 0–9%, mode ¼ 3%) than the Western chimpanzees (range ¼ 0–16%,
mode ¼ 7%).

Population Structure Within the Bossou-Nimba Chimpanzee Group
Table V shows the FST and net (DA) number of nucleotide differencesamong the Bossou population and the two populations of the Nimba Mountains. No population differed significantly from any other population. As in thecase of nucleotide diversity, both minimum and maximum estimates wereconstructed for each population. In all cases in which maximum estimates
were used, negative estimates of DA and FST were obtained. This is because f0 isless than f1 for negative FST, and because p12 is less than (p1+p2)/2 fornegative DA, which means that two genes randomly chosen from two different
populations are more closely related than genes from the same population.
Such a negative value suggests that population differentiation is negligible.
Therefore, the mtDNA analysis does not show any distinct population
structure within the chimpanzee habitat that includes Bossou and the
Nimba Mountains.
We conducted an AMOVA analysis for the Bossou group and the two
Nimba Mountains groups to further investigate their population structures.
Small variances were observed between the Bossou and Nimba Mountaingroups (i.e., Bossou vs. total of two groups of Seringbara and Yealé: s2a ¼ 0.583;
6.27%; P ¼ 0.341; SE ¼ 0.023) and between the two Nimba groups (i.e.,
Seringbara vs. Yealé s2b ¼ 0.251; 2.70%, P ¼ 0.231; SE ¼ 0.015), while thewithin-group variance (s2c ¼ 8.468; 91.03%, P ¼ 0.016; SE ¼ 0.004) constituted
the majority of the total variance (s2T ¼ 9.303). Neither of the between-groupvariance components were statistically significantly different from zero.
Therefore, the AMOVA result also suggests that the mtDNA revealed no
distinct population structure in the chimpanzee habitat of the Bossou-Nimba
region.

TABLE V. Net Number of Nucleotide Differences (DA) and Pairwise FST Among Sampling
SitesnPopulation pair
Bossou vs. Seringbara
Min
Max
Bossou vs. Yealé
Min
Max
Seringbara vs. Yealé
Min
Max

DA

FST

0.0019 (0.077)
0.0010 (0.574)

0.06 (0.076)
0.03 (0.599)

0.0001 (0.367)
0.0005 (0.476)

0.01 (0.334)
0.01 (0.449)

0.0005 (0.283)
0.0030 (0.866)

0.02 (0.234)
0.09 (0.895)

nValues in parentheses are the proportion of permutations leading to a calculated value larger or equal to thatobserved under the null hypothesis of random distribution [Schneider et al., 2000]. Min and Max designate values
calculated using minimum and maximum estimates, respectively, for Seringbara and Yealé population. The real
numbers of variants were used for the Bossou population. See text for detail about minimum and maximum
estimations.

mtDNA of Chimpanzees in Nimba and Bossou / 271
Y-6
P.t.verus1(Dagbego, Ivory Coast)
S-4
Bs-TJFK
P.t.verus2(Liberia)
P.t.verus3(Bossemattie, Ivory Coast)
S-6/ P.t.verus4(Nimba, Ivory Coast)

Cluster 1

Y-7
P.t.verus5(Comoe, Ivory Coast)
S-1
Y-1

76

S-5/P.t.verus6(Solo, Mali)
P.t.verus7(Tai, Ivory Coast)
Y-5
P.t.verus8(Wongo, Mali)
85

Cluster 5

Bs-P

99

96 S-2Cluster 6

Y-3
78 Bs-N
73

Y-8
P.t.verus9(Tai, Ivory Coast)

Cluster 2

P.t.verus10(Solo, Mali)
86
P.t.verus11(Tai, Ivory Coast)
P.t.verus12(Solo, Mali)
Bs-V

Cluster 4

Y-2/P.t.verus13(Tai, Ivory Coast)
P.t.verus14(Comoe, Ivory Coast)

99
69

S-3

Cluster 3

Y-9
Bs-Y
S-7
P.t.vellerosus
89

P.t.shweinfurthii
P.t.troglodytes2

99

97

P.t.troglodytes3
P.t.troglodytes1

0.02

Fig. 3. A neighbor-joining tree of chimpanzee mtDNA variants including those described in thisstudy and previously published data. The root is located using bonobo (Pan paniscus) sequence asan outgroup.

272 / Shimada et al.

Phylogenetic Relationship of mtDNA Variants Within the
Bossou-Nimba Region and Other West African Regions
Figure 3 shows the phylogenetic relationship of the Pan troglodytes verus
mtDNA variants with sequences from the three other subspecies and Pan
panisucus as outgroups. The mitochondrial variants observed in the Bossou
and Nimba regions represent a variety of variants documented within thissubspecies. They do not cluster according to geographic region. In fact, the six
clusters found in Bossou and Nimba region represent all of the major
mitochondrial clades of the verus subspecies. Similar distribution patterns of
mtDNA variants have been reported for West African chimpanzees as well
[Gagneux et al., 1999; Goldberg & Ruvolo, 1997a, b; Morin et al., 1994]. The
absence of a clear population structure of mtDNA variation suggests panmixtureof the ancestral population of the verus subspecies, and that insufficient time
has passed since the separation of the current habitats for geographically distinct
population structure to evolve. These results also suggest that the Bossou-Nimbagroups derived from the ancestral population of the West African chimpanzee
without passing through a serious bottleneck.
DISCUSSION

Relationship Between Sample Size and Number of Mitochondrial
Variants
Figure 4 shows the relationship between the sample sizes and the observed
numbers of variants. This graph was constructed by a random drawing fromour samples. The number of variants found from the Bossou and Seringbara
populations appears to plateau at roughly the number of variants we actually

Seringbara

Yealé

Bossou10

Variant number observed

8

6

4

2

0
0

5

10
15
Sample number examined

20

Fig. 4. Relationship between the number of sequences and the number of variants.

mtDNA of Chimpanzees in Nimba and Bossou / 273

observed, despite the fact that one known variant from the region (Bs-Y) was notfound within the population. In the Yealé population, the number of variants
increased with the sample size. This suggests that the real number of variantsin the Yealé population may be much larger than has been observed thus far.
Limited records of direct observation at Yealé sites suggest the presenceof multiple groups in the area [Humle & Matsuzawa, 2001]. Furthermore,
a bed census study documented many more beds in Yealé than in Seringbara[Shimada, 2000]. This finding supports the expectation of a larger population
size in Yealé.

Variant Sharing Among Bossou and Nimba Groups
Only three mtDNA variants were shared among groups in the Bossou-Nimba
region (Table II). This suggests that there is a low-level gene flow via females inthis region, and seems to contradict the results from the AMOVA analysis. There
are at least two possible explanations for this paradox. The first possibility is
missing variants. According to Fig. 4, we may be missing some mitochondrial
variants in the Yealé population. This may also be true of the Seringbara andBossou populations to some extent. If we conduct more extensive sampling inthese populations, we may be able to find more variants shared among
populations.
Another possibility is that the DA and FST analyses lack sensitivity (seeTable V) for recent population history. These measures may be appropriate forsummarizing the relatively long-term population history, but they may not detect
recent decreases in gene flow. In this case, an examination of shared variants
among populations may be more appropriate for estimating recent population
history. If so, the results shown in Table II suggest that there has been almost no
recent exchange of individuals among the chimpanzee populations in the BossouNimba region.
CONCLUSIONSThis mtDNA survey of chimpanzee populations of the Bossou-Nimba region
revealed two findings: 1) very few mitochondrial variants are shared among thesites sampled, suggesting little gene flow involving mtDNA; and 2) mtDNA
variant distribution does not suggest any clear population structure within the
chimpanzee habitat of the Bossou-Nimba region.
We propose two explanations for these findings: 1) the survey was too limited
to reveal actual gene flow, as suggested by the relationship between the number ofsequences characterized in this study and the number of samples obtained at the
Yealé site; and 2) the ancestral population of Pan troglodytes verus was panmictic,
and insufficient time has passed for a clear pattern in the geographic distributionof mtDNA variants to emerge.
ACKNOWLEDGMENTS
We thank the Institut de Recherche Environmental de Bossou (IREB), theDirection Nationale de la Recherche Scientifique et Technique (DNRST) of
Guinea, and Direction de la Protection de la Nature of Ivory Coast for permission
and support to conduct field research. We acknowledge Drs. T. Matsuzawa, H.
Takemoto, G. Yamakoshi, and M. Myowa-Yamakoshi for helping us to collect the
samples. We thank Dr. O. Takenaka for his advice about collecting and preserving
samples. We also express our thanks to the following field guides: J. Koman,274 / Shimada et al.

G. Goumi, P. Cherif, P. Goumi, J.G. Dore, K. Dore, F. Dore, D. Gray, A. Gogo, F.
Pahon, A. Go-pu, and A. Wanseu. We thank Dr. P. Gagneux for providing
information on his sampling location, and Dr. L. Vigilant and other members ofPrimatology Department at the Max Planck Institute for Evolutionary Anthropology for teaching M.K.S. the DNA extraction technique. We thank Dr. K.E.
Chambers for helpful comments on an earlier version of this manuscript. We
thank Ms. Yoshimi Noaki for her skillful technical assistance, and members of the
Saitou laboratory for helpful discussions. This study was supported by grants-inaid for scientific research from the Ministry of Education, Science, Sports, andCulture, Japan (research on priority area (no. 14011248) to N.S., international
scientific program (no. 10041168) to Y.S., and COE research (no. 10CE2005) tothe Kyoto University Primate Research Institute). M.K.S. was supported during
this study by fellowships from the National Institute of Genetics COE, Nissan
Science Foundation, and Inamori Foundation.

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