Journal of Heredity 2008:99(3):254–264
doi:10.1093/jhered/esn003
Advance Access publication March 11, 2008

! The American Genetic Association. 2008. All rights reserved.
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Mitochondrial DNA Sequence Phylogenyof 4 Populations of the Widely
Distributed Cynomolgus Macaque(Macaca fascicularis fascicularis)
ANTOINE BLANCHER, MAXIME BONHOMME, BRIGITTE CROUAU-ROY, KEIJI TERAO, TAKASHI KITANO, AND
NARUYA SAITOU
From the Laboratoire d’immunogénétique moléculaire, Université Paul Sabatier, Toulouse, France (Blancher); theLaboratoire d’immunologie, Hôpital Rangueil, TSA 50032, 31059 Toulouse cedex 9, France (Blancher); the Laboratoire UMR
CNRS 5174 Evolution et Diversité Biologique, Université Paul Sabatier, Toulouse, France (Bonhomme and Crouau-Roy); thePrimate Centre, National Institute of Infectious Diseases, 1-Hachimandai, Tsukuba, Ibaraki, 305-0843, Japan (Terao); and theDivision of Population Genetics, National Institute of Genetics, Mishima, 411-8540, Japan (Kitano and Saitou). Takashi Kitano
is now at the Faculty of Engineering, Ibaraki University, Hitachi, Japan.
Address correspondence to A. Blancher at the address above, or e-mail: blancher@easynet.fr.

Abstract
We studied the mitochondrial DNA (mtDNA) polymorphism of 304 Macaca fascicularis fascicularis (M. f. fascicularis) individuals,
representative of 4 cynomolgus macaque populations (Indochina, Indonesia, Philippines, and Mauritius). By sequencing
a 590-bp fragment in the hypervariable II region of the D-loop region, we defined 70 haplotypes. The homologous region
was also characterized in 22 Chinese Macaca mulatta and 2 Macaca sylvanus. The phylogenetic analysis confirms the monophylyof M. f. fascicularis and defines 2 haplotype groups inside the M. f. fascicularis clade: one ‘‘insular,’’ encompassing 6 Philippines,
2 Mauritius, and 31 Indonesian haplotypes, the other ‘‘continental’’ that contains all Indochinese and 6 Indonesian
haplotypes. Continental and insular group divergence time was estimated to be approximately 106 years before present (BP).
Among Indonesian haplotypes, some have a continental origin. This suggests either direct migration from mainland toIndonesia or that remnant lineages from an ancient population genetically close to the mainland (i.e., in the Sunda Shelf,,550 000 years BP) were subsequently brought southward to Indonesia. The low nucleotide diversity in the Philippines
population suggests a bottleneck following colonization by Indonesian individuals, around 110 000 years BP. mtDNA andfurther observations of nuclear genetic data corroborate the mixed origin (Indonesian/continental) hypothesis of Mauritius
individuals and a population bottleneck.

Cynomolgus macaque (Macaca fascicularis) is used as a nonhuman primate model for biomedical research in various
domains such as tissue allograft rejection in case of kidney(Borie et al. 2002; Wieczorek et al. 2006) or cardiac(Schroder et al. 2007) transplantation, bone marrow graft(Lau et al. 2004), immune response against pathogens (Satoet al. 2008) or new vaccines (Kita et al. 2005), and infectious
diseases and particularly Simian Immunodeficiency Virus(SIV)-induced AIDS or emergent pathogens (Kuiken et al.
2003; Reed et al. 2005; Lawler et al. 2006). In all thesemodels, the genetic background of the animals could be ofgreat importance; in some cases, it was reported that animal
geographical origin can influence the response to drugs or
the sensibility to the experimental disease (Schmidt et al.
254

1977; Menninger et al. 2002). Numerous reports have been
published on the geographical variations of the cynomolgus
macaque. Blood groups (Terao et al. 1981), blood proteins(Nozawa et al. 1977; Kondo et al. 1993), mitochondrial
DNA (mtDNA) (Harihara et al. 1988; Lawler et al. 1995;
Tosi et al. 2002, 2003; Smith et al. 2007; Tosi and Coke
2007), and Y chromosome DNA (Tosi et al. 2002, 2003;
Tosi and Coke 2007) were studied. The phylogenetic
relationships among the populations of M. fascicularis havebeen discussed, and discrepancies in population divergencetimes between mtDNA and nuclear markers have beenfound. Moreover, the analysis of paleoclimate in South Asia
has led some authors to propose various hypotheses
concerning the geographical dispersal of macaques inBlancher et al. ! Cynomolgus Macaque mtDNA Polymorphism

Southeast Asia (Fooden 1995; Delson 1980; Eudey 1980;
Abegg and Thierry 2002). The cynomolgus macaque ischaracterized by its habitat, the mangroves (Fittinghoff and
Lindburg 1980; Wheatley 1980), which may have favoreda dispersal mode through the estuaries and by sea rafting.
Thus, both habitat characteristics and past sea level changes
in Southeast Asia could explain the wide geographic
distribution of the species. This study focuses on thecommon subspecies Macaca fascicularis fascicularis (M. f.
fascicularis), excluding all other subspecies that are either
restricted to some small islands or morphologically different(i.e., the dark pelage subspecies Macaca fascicularis philippinensis), as extensively described in Fooden (1991, 1995).
Maternally inherited mtDNA has provided valuable datafor investigating intraspecific variation, population structure,phylogeography, and demography in macaque species(Harihara et al. 1988; Melnick and Hoelzer 1992; Lawler
et al. 1995; Tosi et al. 2002; Marmi et al. 2004; Modolo et al.
2005; Smith and McDonough 2005; Kawamoto, Shotake,
et al. 2007; Smith et al. 2007). Harihara et al. (1988), throughrestriction fragment length polymorphism (RFLP) data on
M. fascicularis populations using 5 restriction enzymes applied
to 149 individuals, described 2 population groups: ‘‘insular’’(Philippines and Indonesia) and ‘‘continental’’ (Malaysia andIndochina). The genetic distance between the haplotypes ofthese 2 groups suggests a deep split between the continental
and the insular groups. The dichotomy between insular andcontinental populations was confirmed by Tosi et al. (2002,
2003) and Tosi and Coke (2007). However, the mtDNA
lineage heterogeneity found for the Indonesian populationsuggests a complex origin.
Lawler et al. (1995) showed that cynomolgus macaquefrom Mauritius have only a few mitochondrial RFLP
haplotypes, all possibly deriving from a single one. Thisobservation indicates a recent founding of the Mauritius
macaque population by an artificial introduction between 300
and 500 years ago (see Sussman and Tattersall 1986). Sailors
imported a few animals most probably from Indonesia, asconfirmed by the genetic proximity of 1) Mauritius and Java
populations using nuclear and mtDNA markers (Kondo et al.
1993; Kawamoto Y, Kawamoto S, et al. 2008, Tosi and Coke
2007) and of 2) Mauritius and Sumatra by means of Y
chromosome sequences (Tosi and Coke 2007).
In the present study, we investigate the molecular genetic
relationships between cynomolgus macaque populations andone rhesus macaque (Macaca mulatta) population, using
mtDNA sequence data. The aim of this study is to refine
more precisely the phylogeographic structure of the cynomolgus macaque, to make inferences about the dispersal
scenario of this species, and to confront mtDNA polymorphism to nuclear genetic diversity (Bonhomme et al.
2007) in the same samples. Herein, we sequenced around 590
bp in hypervariable segment 2 (HVII) of the D-loop of a large
number of individuals (304) from 4 cynomolgus macaquepopulations (Indochina, Indonesia, Philippines, and Mauritiusisland). The artificially established Mauritius population
was studied to trace the most probable ancestral populationand the signature of the founding effect. The phylogenetic

relationships of the 3 natural populations (Indonesia, Indochina, and Philippines) are discussed with regard to thecolonization of Southeast Asia by M. f. fascicularis.

Materials and Methods
Animals and DNA Samples
Genomic DNA was extracted from peripheral blood of 304
unrelated M. f. fascicularis using either QIA amp Blood Kit(Qiagen, Courtaboeuf, France) or phenol-chloroform extraction. DNA blood samples of 137 animals captured fromthe Indonesian wild population were obtained from the
Tsukuba Primate Center through K.T. Other animals are F1
derived from breeders that had been captured in the wild on
various locations in Mauritius (N 5 74), Philippines (N 525), and Indochina (N 5 68) by Noveprim (Mauritius),
Siconbrec (Philippines), and Nafovanny (Vietnam), respectively. Macaca mulatta blood samples (N 5 22) were obtained
from the Bioprim quarantine center (Bioprim, Baziège,
France). The rhesus monkeys were F1 imported froma Chinese breeding center (South China Primate Research
Development Center, Jiufu center, Guangzhou, China).
Macaca sylvanus blood samples from 2 unrelated individuals
were provided by Dr. Ducos de Lahitte (Ecole NationaleVétérinaire, Toulouse, France).
Polymerase Chain Reaction and Sequencing
A 590-bp fragment of the HVII of the mtDNA D-loop was
amplified by using the pair of primers: Saru-4F (5#ATCACGGGTCTATCACCCTA-3#) and Saru-5R (5#GGCCAGGACCAAGCCTATTT-3#). This primer pair has
been successfully used for Japanese macaque mtDNA studies(Hayasaka et al. 1991; Kawamoto, Shotake, et al. 2007) and isalso known to produce no amplification of nuclear-inserted
mtDNA sequences in M. f. fascicularis (Dr. Kawamoto Y,
personal communication). This pair amplifies positions 22–606
in the complete sequence of M. mulatta mitochondrial genome(DNA Databank of Japan-DDBJ/European Molecular Biology
Laboratory-EMBL/GenBank accession number AY612638).
Amplified material was separated on agarose gels, purified
by using QIAquick PCR Purification Kit (Qiagen, Courtaboeuf, France), and directly sequenced on both strands by thefluorescent Dye Terminator method on an ABI 373 automaticsequencer (Applied Biosystems Japan, Tokyo, Japan) or an
CEQ 2000 automatic sequencer (Beckman Coulter, Paris,
France). In all population samples, we paid special attention tosequences differing from the most frequent and checked allthese sequences by sequencing again the target fragment.
Sequence Data Analysis
The M. f. fascicularis sequences characterized were alignedwith macaque mtDNA sequences obtained from 2 M.
sylvanus and 22 M. mulatta (this study) and homologous
sequences available in DDBJ/EMBL/GenBank International Nucleotide Sequence Database (2 Macaca fuscata and 1
Papio hamadryas, accession numbers M80209, M80210, and255

Journal of Heredity 2008:99(3)Y18001). Nucleotide diversity p (Nei 1987), Tajima’s D(Tajima 1989), and Fu’s (1997) Fs were calculated using the
ARLEQUIN ver. 2.000 package (Schneider et al. 2000) on
the HVII region dataset reported here and on the HVI
region dataset of Smith et al. (2007). We constructed
phylogenetic trees, based on 590 nucleotides, using 3
different methods. First, a neighbor-joining tree (Saitou
and Nei 1987) was constructed with the Kimura’s 2parameter distance (Kimura 1980), using the computer
program MEGA ver. 3.0 (Kumar et al. 2004), and 10 000
bootstraps were performed to determine the significance oftree branching. We then performed maximum likelihood(ML) and Bayesian phylogenetic analyses using the most
likely substitution model inferred using a likelihood framework implemented in the software MODELTEST 3.7(Posada and Crendall 1998). This software tests 56 different
substitution models and estimates the most likely one using
a likelihood ratio test. ML inference was performed using
the PHYML software (Guindon and Gascuel 2003), and
10 000 bootstraps were performed on the ML tree. Finally,a Bayesian analysis was performed using the software
MrBayes (Ronquist and Huelsenbeck 2003), where 2
Markov Chains were run on 10 " 106 generations witha sampling each 100 generations. Such a run length allowed
the standard deviation of allelic frequencies to pass below
0.01 and the potential scale reduction factor to reach a valueof 1, as suggested by the authors. The first 25 000 trees(25%) were discarded from the analysis as a burn-in.

Results

Macaca fascicularis fascicularis mtDNA Sequence Diversity
In total, 120 positions are polymorphic in 304 individuals,
including 8 positions with indels (Figure 1), for a total of 70
haplotypes. Among the 112 positions with substitutions, 109
are dimorphic with transitions being much more frequent(65 C 4 T and 38 A 4 G) than transversions (6 in total).
Only 2 populations, Indonesia and Mauritius, share one
haplotype that is present in all Mauritius animals but one
and in 6 out of 137 animals from Indonesia.
The intrapopulation diversity is shown in Table 1(a).
Nucleotide diversity (p) was the highest (0.032) for
Indonesia, followed by Indochina (0.017) and Philippines
population (0.009). The Mauritius population has only 2
haplotypes differing by a single-base substitution. Accordingly, the nucleotide diversity (4.6 " 10#5) was extremely
low for this population. The gene diversity (h) in Indochina
and Indonesia is similar in our study (HVII) and in that ofSmith et al. (2007) based on HVI, as shown in Table 1 (a, b).
However, gene diversity is higher with HVI than with HVII
in Philippines and Mauritius. The nucleotide diversity is atleast 5 times higher for HVI than for HVII in all
populations. This confirms, in M. f. fascicularis, the higher
mutation rate in the HVI region than in the HVII region of
the D-loop, found in humans (Meyer et al. 1999). Except for
Mauritius, both neutrality tests for HVI and HVII revealthat polymorphism is neutral, as expected for the mtDNA
D-loop region. In the case of the Mauritius population, only
HVI (and not HVII) analysis revealed significantly negative
FS and D values. The greater polymorphism of HVI may
thus indicate a recent expansion in the Mauritius population.

Determination of mtDNA Sequences

Phylogenetic Analysis

Primate mtDNA is known to be often inserted into nuclear
DNA—‘‘numts’’—(Bensasson et al. 2001). Numts wereshown to hinder the evolutionary history inference ofprimate species (Thalmann et al. 2004, 2005). Thus, we
carefully considered the putative amplification and sequencing of numts, although caution was done when choosing thepolymerase chain reaction (PCR) primers (see Materials andMethods). With all M. fascicularis samples, we obtained a single
band by PCR amplification, and most direct sequencing
results showed unique nucleotide sequence for each individual. As for M. mulatta samples, about 1/3 of PCR
products did not show unique sequence, so we chose ones
that were very close to the complete sequence of M. mulatta
mitochondrial genome (DDBJ/EMBL/GenBank accession
number AY612638). Our 2 newly determined M. sylvanus
sequences were also compared with the complete sequenceof M. sylvanus mitochondrial genome (DDBJ/EMBL/
GenBank accession number AJ309865), and they were foundto be very close with each other. Furthermore, phylogenetic
analyses of these sequences produced trees consistent withthe known macaque phylogeny (see below). In conclusion,
we believe that our nucleotide sequences were derived fromauthentic mtDNA. Nucleotide sequences determined in thisstudy were deposited to the DDBJ/EMBL/GenBank underaccession numbers AB261898–AB261974.

The neighbor-joining, ML, and Bayesian trees have the same
topology. We thus show only the Bayesian tree (Figure 2).
All the M. f. fascicularis sequences clustered together(posterior value 5 91%), showing that M. f. fascicularis ismonophyletic.
Inside the M. f. fascicularis clade, 2 main groups ofsequences can be detected. Group I encompasses all
haplotypes from the Philippines and Mauritius samples
and most of the Indonesian sample haplotypes (29 out of
35). The sequences from the Philippines population and thatfrom one Indonesian individual constitute a subgroup I-1(posterior value 5 85%). There are 3 other subclusters ingroup I (I-2, I-3, and I-4), all supported with posteriorvalues higher than 77% (see Figure 2). Two Mauritius
haplotypes belong to subcluster I-3 with 14 Indonesian
haplotypes (posterior value 5 98%), and the remaining 3
subclusters (I-2 and I-4) consist of 2 and 12 Indonesian
haplotypes, respectively. Group II includes all Indochinese
haplotypes and 6 haplotypes from Indonesia, and we
divided it into 3 subclusters (II-1, II-2, and II-3), as shownin Figure 2. Subclusters II-1 and II-3 include haplotypes
only found in Indochina (2 and 25, respectively), whereas
subcluster II-2 consists of 6 Indonesian haplotypes that arecloser to Indochinese haplotypes than to Indonesian
haplotypes of group I (posterior value 5 97%).

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Blancher et al. ! Cynomolgus Macaque mtDNA Polymorphism

Figure 1. Variant sites of the mtDNA control region (HVII) in Macaca fascicularis fascicularis determined in this study. The
sequence names begin with letters corresponding to the 4 populations (InCH 5 Indochina; InNE 5 Indonesia; Mau 5 Mauritius;and Phi 5 Philippines), followed by a code number identifying the sequence and a number in brackets corresponding to thenumber of animals sharing the sequence. Stars in the fourth line indicate the positions that differentiate Indochinese sequencesfrom those of other populations.

Dating the Divergence of Macaca fascicularis fascicularis
Populations
The Bayesian tree showing the length of branches (Figure 2)
was transformed under the assumption of a constant rate of
mutation, while keeping its tree topology, and the resulting
tree is shown in Figure 3. In the absence of M. f. fascicularis
fossil data allowing us to date interpopulational divergences,

calibration of the tree is difficult. Therefore, we decided tocalibrate it at node 0, which separates M. fascicularis and M.
mulatta clusters. Among all the dates proposed, we chose
1.6 million years before present (My BP) as the mostprobable divergence time between the 2 macaque species.
This divergence time was deduced from a composite
estimate of primate divergence times (Purvis 1995). In that257

Journal of Heredity 2008:99(3)Table 1.

Summary statistics for mtDNA and nuclear genetic diversity in Macaca fascicularis fascicularis populations(a) Based on HVII region sequences observed in 4 M. f. fascicularis populations (this study)
Diversity parameters

Neutrality tests

Population

Nhap (poly sites/indel)p " 1000 (±SE)

h (±SE)

D (P value)

Fs* (P value)

Indochina (68)
Indonesia (137)Philippines (25)
Mauritius (74)27 (59/6)
35 (88/4)
6 (16/0)
2 (1/0)17.1 (±8.7)32.4 (±16)
8.85 (±4.93)
0.046 (±0.164)

0.932 (±0.016)
0.926 (±0.013)
0.693 (±0.082)
0.027 (±0.026)#0.33 (0.42)
0.58 (0.21)
0.82 (0.18)#1.06 (0.11)#3.14 (0.18)
1.68 (0.28)
3.66 (0.07)#1.97 (0.04)(b) Based on HVI region sequences observed in 4 M. f. fascicularis populations (Smith et al. 2007)
Diversity parameters

Neutrality tests

Population

Nhap (poly sites)p " 1000 (±SE)

h (±SE)

D (P value)

Fs* (P value)

Indochina (89)Indonesia (70)Philippines (83)
Mauritius (68)

48 (107)58 (179)
34 (124)
9 (15)

83.9 (±41.4)
183.7 (±89.3)
59.5 (±29.8)
2.9 (±2.4)0.975 (±0.0064)
0.994 (±0.0035)
0.78 (±0.05)
0.298 (±0.073)#0.04 (0.57)
0.80 (0.16)#1.36 (0.067)#2.24 (0.001)#6.29 (0.12)#10.39 (0.03)#2.23 (0.31)#5.21 (0.005)(c) Based on the microsatellite dataset from Bonhomme et al. (2007)
Diversity parameters

Diversity parameters

MHC

non-MHC

Population

nA

He

nA

He

Indochina (70)Java (38)Philippines (65)
Mauritius (81)

12
11
8
8

0.80
0.76
0.68
0.70

12
9
8
7

0.81
0.83
0.74
0.69

The number of individuals studied follows the population name; Nhap 5 Numbers of haplotypes in each population; poly sites 5 number of polymorphic
sites; indel: number of insertion–deletions in each population sample; p 5 mean number of pairwise differences; SE 5 standard error for the sampling
process; h 5 gene diversity; D 5 Tajima’s D; FS 5 Fu’s FS; nA 5 mean number of alleles; and He 5 mean expected heterozygosity.
* Fu’s FS should be considered as significant at the 5% level if P , 0.02. NB: MHC and non-MHC datasets each consists of 7 microsatellite markers.

study, the baboon–macaque divergence time was proposedat around 9.25 My BP (mean value), a date compatible withmore recent estimates (Raaum et al. 2005; Steiper and Young2006). Moreover, the 1.6 My estimate for M. mulatta and M.
fascicularis divergence time is in the range of dates proposed
by Hayasaka et al. (1996) for the radiation of Asian
macaques. With node 0 of Figure 3 being calibrated at 1.6
My BP, nodes 1, 2, and 3 become 1.2, 0.55, and 0.11 My BP,
respectively. The divergence observed at node 1 corresponds
to the separation between groups I and II, namely between
the continental and insular groups of M. f. fascicularis, whereas
nodes 2 and 3 correspond to the divergence time ofIndochinese and Philippines populations, respectively.

Discussion
Monophyly of Macaca fascicularis fascicularis
This study, based on 4 M. f. fascicularis populations, clearly
confirms that this subspecies is supported as a monophyletic
mtDNA lineage by reference to other macaques (M. mulatta,
M. fuscata, and M. sylvanus). This is in accordance with the
mtDNA phylogenies of macaque species in Tosi et al. (2002,
2003) and Smith et al. (2007). In contrast to mtDNA, theparaphyly of sequences from the Y chromosome andautosomal genes led to suspect a possible male introgression
from M. mulatta to M. fascicularis in Indochina (Tosi et al.
2002, 2003). Macaca nemestrina is also distributed in!

Figure 2. Bayesian phylogenetic tree of mitochondrial sequences of Macaca fascicularis fascicularis, with branch length. The
sequence names begin by letters corresponding to the 4 populations (InCH 5 Indochina; InNE 5 Indonesian; Mau 5 Mauritius;and Phi 5 Philippines), followed by a code number identifying the sequence. When there is no number in brackets the sequence
was observed in only one animal. Numbers at nodes of the tree correspond to posterior confidence values. Sequences from othermacaque species (Macaca sylvanus, Macaca mulatta, and Macaca fuscata) are included in the tree, rooted with a baboon (Papio hamadryas)
sequence (the DDBJ/EMBL/GenBank accession number Y18001) as outgroup. The 2 M. fuscata sequences in this tree were from
Hayasaka et al. (1991), and all other sequences were newly determined in this study.

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Blancher et al. ! Cynomolgus Macaque mtDNA Polymorphism

259

Journal of Heredity 2008:99(3)Indonesia, as well as M. fascicularis and this could again bea possible source of introgression. Therefore, when inferring
the phylogenetic relationships of macaque species, caution
should be taken as for the choice of the molecular markers
and for the sampling of species and populations.
Origin and Genetic Diversity of the Mauritius MacaquePopulation
Among the 4 populations studied here, that from Mauritius
clearly shows distinct polymorphism patterns. We foundthat most animals (73 out of 74) have the same sequence,
whereas the remaining animal differs by only one nucleotide
position. The latter sequence was validated twice by
sequencing the DNA obtained from 2 independent PCRs.
In 6 animals of the Indonesian sample, we found sequences
strictly identical to the most frequent Mauritius haplotype,confirming that Indonesia is a very probable source of theMauritius population founders. This result is compatible
with the Javanese origin previously suggested by different
studies (Sussman and Tattersall 1981; Kondo et al. 1993;
Kawamoto Y, Kawamoto S, et al. 2008). However, recently,
Tosi and Coke (2007) clearly demonstrated a mixed originof the Mauritius population, with a Javanese or Sumatranmaternal lineage, and a continental Y chromosome lineage
currently found in Sumatra. In the same way, recent results
obtained on DRB exon 2 polymorphism show that theMauritius population shares several DRB sequences withnot only Indonesian but also Indochinese cynomolgus
macaques (9 and 6 sequences, respectively), and only 2
sequences with the Philippines (Blancher et al. 2006, andadditional unpublished data from A. Blancher). Thus, thestudy of Tosi and Coke (2007) and our observations lead topropose the mixed origin of the Mauritius population as themost probable hypothesis.
Most probably, the low-frequency Mauritius haplotype(1%), not observed in the Indonesian sample, may havearisen by mutation (T / C) from the main haplotype, afterM. f. fascicularis became established in Mauritius 300–500
years ago. These results are in agreement with the study of
Kawamoto Y, Kawamoto S, et al. (2008) who described 2
haplotypes (S and L) of an HVII fragment sequenced on thesame number of individuals, with the low-frequency
haplotype at 9%. The extremely low nucleotide and genetic
diversities found in the Mauritius sample is most probably
the current molecular signature of a founding effect, withfew females, that was firstly deduced by Lawler et al. (1995)
using RFLP. Our results, which clearly demonstrate that theMauritius population is much less polymorphic (h 5 0.03)
than the Philippines one (h 5 0.69) for mtDNA, differ fromthose recently observed for DRB exon 2 polymorphism inthese 2 insular populations (Blancher et al. 2006). From thisDRB dataset, the expected heterozygosity values weresimilar between the Mauritius (h 5 0.78) and Philippines

samples (h 5 0.81). In addition, genetic data from otherstudies of the nuclear genome emphasize the discrepancy
observed between mtDNA and nuclear genomes in
Mauritius: Average He from microsatellite data equaled
0.66 (Kawamoto Y, Kawamoto S, et al. 2008), 0.70, and 0.69
for MHC and non-MHC microsatellite data (Table 1c,
Bonhomme et al. 2007). This discrepancy between mtDNA
and nuclear (microsatellite and DRB) markers in Mauritius
could have several levels of explanation: 1) the effective size
(Ne) of the mtDNA is 1/4 the nuclear genes, so theprobability for the Mauritius population to retain and/or
maintain polymorphism after a recent founding event(;100 generations) is much lower for mtDNA than for
nuclear genes; 2) the approximate date of the Philippines
foundation is very much older than that of Mauritius (0.11
My BP instead of 400 years BP) and after such a long period
following the colonization, mtDNA polymorphism in thePhilippines population had enough time to be restored; and3) a biased sex ratio toward males in the Mauritius founders
may explain the rapid loss of mtDNA lineages. Under thishypothesis, the male founders (continental/Sumatran origin,
Tosi and Coke 2007) could have strongly contributed to
Mauritius cynomolgus macaque nuclear polymorphism.
Dispersal Scenario of Macaca fascicularis fascicularis
Populations
Based on the mtDNA sequence analysis of 3 natural M. f.
fascicularis populations, we propose the following dispersal
scenario (see Figure 4). First, the ancestral population of M.
f. fascicularis diverged (see also Harihara et al. 1988) into 2
subpopulations: one called continental, colonizing the
Indochinese peninsula and the other, called insular, almost
1 My BP. This estimate is close to that proposed by Tosi
et al. (2003; see also Tosi and Coke 2007).
A more precise past history of the cynomolgus macaqueis given by the phylogenetic tree: 6 Indonesian haplotypes(group II-2 of Figure 2) cluster in the continental group of
sequences (group II of Figure 2). This suggests the current
presence in Indonesia of mtDNA lineages coming directlyfrom the Indochinese peninsula via migrant individuals.
Alternatively, those lineages may originate from a remnant
population that was localized in the Sunda Shelf and closely
connected to the Indochinese peninsula, during the complex
dynamics of sea level change in this region at Pleistocene
glacial periods (,550,000 years BP). Figure 4 shows theputative location on the Sunda Shelf of such an ancient
population and gives possible migrations routes explaining
the paraphyly of Indonesian haplotypes.
More recently, around 110 000 years BP, the Philippines
population derived from an Indonesian M. f. fascicularis stock(see Figure 3). Invasion of the Philippines may have beenpossible by terrestrial access through Borneo during periodsof low sea level in Southeast Asia. An alternative scenario is!

Figure 3. Bayesian phylogenetic tree of Macaca fascicularis populations, under the assumption of a constant mutation rate. Circles
represent the nodes 0, 1, 2, and 3 that are dated at 1.6, 1.2, 0.55, and 0.11 My, respectively.
260

Blancher et al. ! Cynomolgus Macaque mtDNA Polymorphism

261

Journal of Heredity 2008:99(3)Figure 4. Map depicting the dispersal of Macaca fascicularis fascicularis in Southeast Asia. The figure was adapted from the original
map from Voris (2000) where the limits of #120 m below present sea level are shown in light gray. Solid arrows depict the initial
split between continental and insular groups and the colonization event of the Philippines. Dashed arrows depict hypothetic
dispersal subsequent to the initial split. Dotted arrows represent the putative origins of the Mauritius population.

that, given that M. fascularis live in mangroves near the coast,
it could have colonized the Philippines from Indonesia via
Borneo by sea rafting (Abegg and Thierry 2002).
Most probably, all current Philippines haplotypes
reported here have derived from a single common ancestral
haplotype. The low genetic and nucleotide diversities
suggest that a small group of Indonesian animals colonized
the Philippines. Although precise dating of Philippines
colonization cannot be assessed from our results, thephylogenetic tree suggests that after colonization of thePhilippines, the population essentially remained isolated
from the Indonesian population, allowing divergence ofPhilippines haplotypes that are not observed in theIndonesian sample. Although most Philippines sequences
cluster in a specific group, this group encompasses one
sequence from Indonesia. The latter could derive from thesame ancestral haplotype, which gave rise to the Philippines
population, but we cannot exclude back migration from thePhilippines to Indonesia (Harihara et al. 1988).

Conclusions
The analysis of mtDNA D-loop sequences and further
observations of nuclear genetic data confirms the foundingof the Mauritius population by a small number of animals
imported from Indonesia and perhaps also Indochina, aspreviously suggested by Tosi and Coke (2007). Our results
also support the previously suggested ancient differentiationof M. f. fascicularis into 2 subgroups: 1 continental, anotherinsular. In addition, our data raise the complexity of theIndonesian population. Indeed, the similarity between somesequences from Indonesia (subgroup II-2) and Indochina
suggests either subsequent migrations southward, from262

Indochina to Indonesia, or an early colonization of the
Sunda Shelf by the Indochinese population and thenmigrations southward, to Indonesia. The Philippines weremost probably colonized by a small number of IndonesianM. f. fascicularis individuals. Unfortunately, the paucity of M.
f. fascicularis fossil data in South East Asia and the uncertainty
in calibrating the phylogenetic tree make divergence times
dating difficult to assess in the light of complex and periodic
changes in paleoclimates in Southeast Asia.

Funding
French Ministry of Research grant (contract EA3034 toA.B); Japanese Ministry of Education, Sport, Science, andTechnology (to N.S.).

Acknowledgments
We thank, for their excellent technical assistance, Stéphanie Despiau,
Béatrice Atlan, and Yoshimi Noaki. We also thank Dr. Jeffry Rogers for
informing us of the paper of Smith et al. before publication and Dr. DavidGlenn Smith for sending us the geographical origins of all the cynomolgus
macaque individuals they studied.

References
Abegg C, Thierry B. 2002. Macaque evolution and dispersal in insular
south-east Asia. Biol J Linn Soc. 75:555–576.
Bensasson D, Zhang D, Hartl DL, Hewitt GM. 2001. Mitochondrial
pseudogenes: evolution’s misplaced witnesses. Trends Ecol Evol.
16:314–321.
Blancher A, Tisseyre P, Dutaur M, Apoil PA, Maurer C, Quesniaux V, Raulf
F, Bigaud M, Abbal M. 2006. Study of Cynomolgus monkey (Macaca
fascicularis) MhcDRB (Mafa-DRB) polymorphism in two populations.
Immunogenetics. 58:269–282.

Blancher et al. ! Cynomolgus Macaque mtDNA Polymorphism
Bonhomme M, Blancher A, Jalil FM, Crouau-Roy B. 2007. Factors shaping
genetic variation in the MHC of natural non-human primate populations.
Tissue Antigens. 70:398–411.

Lau M, Vayntrub T, Grumet FC, Lowsky R, Strober S, Hoppe R, Larson M,
Holm B, Reitz B, Borie D. 2004. Short tandem repeat analysis to monitor
chimerism in Macaca fascicularis. Am J Transplant. 4:1543–1548.

Borie D, Hausen B, Larson M, Klupp J, Stalder M, Birsan T, Morris R.
2002. A life-supporting technique of renal allotransplantation in Macaca
fascicularis to evaluate novel immunosuppressive drugs in nonhumanprimates. J Surg Res. 107:64–74.

Lawler JV, Endy TP, Hensley LE, Garrison A, Fritz EA, Lesar M, Baric RS,
Kulesh DA, Norwood DA, Wasieloski LP, et al. 2006. Cynomolgus
macaque as an animal model for severe acute respiratory syndrome. PLoS
Med. 3:e149.

Delson E. 1980. Fossil macaques, phyletic relationships and a scenario of
deployment. In: Lindburg DG, editor. The macaques: studies in ecology,
behavior and evolution, New York: Van Nostrand Reinhold Company.
p. 10–30.

Lawler SH, Sussman RW, Taylor LL. 1995. Mitochondrial DNA of theMauritian macaques (Macaca fascicularis): an example of the founder effect.
Am J Phys Anthropol. 96:133–141.

Eudey A. 1980. Pleistocene glacial phenomena and the evolution of Asian
macaques. In: Lindburg DG, editor. The macaques: studies in ecology,
behavior and evolution, New York: Van Nostrand Reinhold Company.
p. 52–83.
Fittinghoff NA, Lindburg DG. 1980. Riverine refuging in east bornean
Macaca fascicularis. In: Lindburg DG, editor. The macaques: studies inecology, behavior and evolution, New York: Van Nostrand Reinhold
Company. p. 215–245.
Fooden J. 1991. Systematic review of Philippine macaques (primates,
cercopithecidae: Macaca fascicularis subspp). Field Zool. 64:1–44.
Fooden J. 1995. Systematic review of Southeast Asian longtail macaques,
Macaca fascicularis [Raffles, (1821)]. Field Zool. 81:1–206.
Fu YX. 1997. Statistical tests of neutrality against population growth,
hitchhiking and background selection. Genetics. 147:915–925.
Guindon S, Gascuel O. 2003. A simple, fast, and accurate algorithm toestimate large phylogenies by maximum-likelihood. Syst Biol. 52:696–704.
Harihara S, Saitou N, Hirai M, Aoto N, Terao K, Cho F, Honjo S, Omoto
K. 1988. Differentiation of mitochondrial DNA types in Macaca fascicularis.
Primates. 29:117–127.
Hayasaka K, Fujii K, Horai S. 1996. Molecular phylogeny of macaques:
implications of nucleotide sequences from an 896-base pair region of
mitochondrial DNA. Mol Biol Evol. 13:1044–1053.
Hayasaka K, Ishida T, Horai S. 1991. Heteroplasmy and polymorphism in the
major noncoding region of mitochondrial DNA in Japanese monkeys:
association with tandemly repeated sequences. Mol Biol Evol. 8:399–415.
Kawamoto Y, Kawamoto S, Matsubayashi K, Nozawa K, Watanabe T,
Stanley M-A, Perwitasari-Farajallah D. 2008. Genetic diversity of longtail
macaques (Macaca fascicularis) on the island of Mauritius: an assessment of
nuclear and mitochondrial DNA polymorphisms. J Med Primatol.
37:45–54.
Kawamoto Y, Shotake T, Nozawa K, Kawamoto S, Tomari K, Mawai S,
Shiri K, Morimitsu Y, Takagi N, Akaza H, et al. 2007. Postglacial
population expansion of Japanese macaques (Macaca fuscata) inferred frommitochondrial DNA phylogeography. Primates. 48:27–40.
Kimura M. 1980. A simple method for estimating evolutionary rates of basesubstitutions through comparative studies of nucleotide sequences. J Mol
Evol. 16:111–120.
Kita Y, Tanaka T, Yoshida S, Ohara N, Kaneda Y, Kuwayama S, Muraki Y,
Kanamaru N, Hashimoto S, Takai H, et al. 2005. Novel recombinant BCG
and DNA-vaccination against tuberculosis in a cynomolgus monkey model.
Vaccine. 23:2132–2135.
Kondo M, Kawamoto Y, Nozawa K, Matsubayashi K, Watanabe T, Griffiths
O, Stanley MA. 1993. Population genetics of crab-eating macaques (Macaca
fascicularis) on the island of Mauritius. Am J Primatol. 29:167–182.
Kuiken T, Rimmelzwaan GF, Van Amerongen G, Osterhaus AD. 2003.
Pathology of human influenza A (H5N1) virus infection in cynomolgus
macaques (Macaca fascicularis). Vet Pathol. 40:304–310.
Kumar S, Tamura K, Nei M. 2004. MEGA3: integrated software formolecular evolutionary genetics analysis and sequence alignment. Brief
Bioinform. 5:150–163.

Marmi J, Bertranpetit J, Terradas J, Takenaka O, Domingo-Roura X. 2004.
Radiation and phylogeography in the Japanese macaque, Macaca fuscata. Mol
Phylogenet Evol. 30:676–685.
Melnick DJ, Hoelzer GA. 1992. Differences in male and female macaquedispersal lead to contrasting distributions of nuclear and mitochondrial
DNA variation. Int J Primatol. 13:379–393.
Menninger K, Wieczorek G, Riesen S, Kunkler A, Audet M, Blancher A,
Schuurman HJ, Quesniaux V, Bigaud M. 2002. The origin of cynomolgus
monkey affects the outcome of kidney allografts under Neoral immunosuppression. Transplant Proc. 34:2887–2888.
Meyer S, Weiss G, von Haeseler A. 1999. Pattern of nucleotide substitution
and rate heterogeneity in the hypervariable regions I and II of human
mtDNA. Genetics. 152:1103–1110.
Modolo L, Salzburger W, Martin RD. 2005. Phylogeography of barbary
macaque (Macaca sylvanus) and the origin of the Gibraltar colony. Proc Natl
Acad Sci USA. 102:7392–7397.
Nei M. 1987. Molecular evolutionary genetics. New York: Columbia
University Press.
Nozawa K, Shotake T, Ohkura Y, Tanabe Y. 1977. Genetic variations
within and between species of Asian macaques. Jpn J Genet. 52:15–30.
Posada D, Crandall KA. 1998. MODELTEST: testing the model of DNA
substitution. Bioinformatics. 14:817–818.
Purvis A. 1995. A composite estimate of primate phylogeny. Philos Trans R
Soc Lond B Biol Sci. 348:405–421.
Raaum RL, Sterner KN, Noviello CM, Stewart CB, Disotell TR. 2005.
Catarrhine primate divergence dates estimated from complete mitochondrial genomes: concordance with fossil and nuclear DNA evidence. J Hum
Evol. 48:237–257.
Reed DS, Larsen T, Sullivan LJ, Lind CM, Lackemeyer MG, Pratt WD, Parker
MD. 2005. Aerosol exposure to western equine encephalitis virus causes
fever and encephalitis in cynomolgus macaques. J Infect Dis. 192:
1173–1182.
Ronquist F, Huelsenbeck JP. 2003. MrBayes 3: Bayesian phylogenetic
inference under mixed models. Bioinformatics. 19:1572–1574.
Saitou N, Nei M. 1987. The neighbor-joining method: a new method forreconstructing phylogenetic trees. Mol Biol Evol. 4:406–425.
Sato H, Kobune F, Ami Y, Yoneda M, Kai C. 2008. Immune responses
against measles virus in cynomolgus monkeys. Comp Immunol Microbiol
Infect Dis. 31:25–35.
Schmidt LH, Fradkin R, Harrison J, Rossan RN. 1977. Differences in thevirulence of Plasmodium knowlesi for Macaca irus ( fascicularis) of Philippine
and Malayan origins. Am J Trop Med Hyg. 26(4):612–622.
Schneider S, Roessli D, Excoffier L. 2000. Arlequin ver. 2000: a software
for population genetics data analysis. Geneva (Switzerland): Genetics andBiometry Laboratory, University of Geneva.
Schroder C, Pierson RN 3rd, Nguyen BN, Kawka DW, Peterson LB, Wu
G, Zhang T, Springer MS, Siciliano SJ, Iliff S, et al. 2007. CCR5 blockade
modulates inflammation and alloimmunity in primates. J Immunol. 179:
2289–2299.

263

Journal of Heredity 2008:99(3)Smith DG, McDonough J. 2005. Mitochondrial DNA variation in
Chinese and Indian rhesus macaques (Macaca mulatta). Am J Primatol. 65:1–25.
Smith DG, McDonough JW, George DA. 2007. Mitochondrial DNA
variation within and among regional populations of longtail macaques(Macaca fascicularis) in relation to other species of the fascicularis group of
macaques. Am J Primatol. 69:182–198.
Steiper ME, Young NM. 2006. Primate molecular divergence dates. Mol
Phylogenet Evol. 41:384–394.
Sussman RW, Tattersall I. 1981. Behavior and ecology of Macaca fascicularis
in Mauritius: a preliminary study. Primates. 22:192–205.
Sussman RW, Tattersall I. 1986. Distribution, abundance, and putative
ecological strategy of Macaca fascicularis on the island of Mauritius,
southwestern Indian Ocean. Folia Primatol. 46:28–43.
Tajima F. 1989. Statistical method for testing the neutral mutation
hypothesis by DNA polymorphism. Genetics. 123:585–595.
Terao K, Fujimoto K, Cho F, Honjo S. 1981. Inheritance and distribution ofhuman-type A-B-O blood groups in cynomolgus monkeys. J Med Primatol.
10:72–80.
Thalmann O, Hebler J, Poinar HN, Paabo S, Vigilant L. 2004. Unreliable
mtDNA data due to nuclear insertions: a cautionary tale from analysis ofhumans and other great apes. Mol Ecol. 13:321–335.
Thalmann O, Serre D, Hofreiter M, Lukas D, Eriksson J, Vigilant L. 2005.
Nuclear insertions help and hinder inference of the evolutionary history ofgorilla mtDNA. Mol Ecol. 14:179–188.

264

Tosi AJ, Coke CS. 2007. Comparative phylogenetics offer new insights into
the biogeographic history of Macaca fascicularis and the origin of theMauritian macaques. Mol Phyl Evol. 42:498–504.
Tosi AJ, Morales JC, Melnick DJ. 2002. Y-chromosome and mitochondrial
markers in Macaca fascicularis indicate introgression with Indochinese
M. mulatta and a biogeographic barrier in the Isthmus of Kra. Int J
Primatol. 23:161–178.
Tosi AJ, Morales JC, Melnick DJ. 2003. Paternal, maternal, and biparental
molecular markers provide unique windows onto the evolutionary historyof macaque monkeys. Evolution. 57:1419–1435.
Voris HK. 2000. Maps of Pleistocene sea levels in Southeast Asia:
shorelines, river systems and time durations. J Biogeogr. 27:1153–1167.
Wheatley BP. 1980. Feeding and ranging of East Bornean Macaca fascicularis.
In: Lindburg DG, editor. The macaques: studies in ecology, behavior
and evolution, New York: Van Nostrand Reinhold Company. p. 215–245.
Wieczorek G, Bigaud M, Menninger K, Riesen S, Quesniaux V, Schuurman
HJ, Audet M, Blancher A, Mihatsch MJ, Nickeleit V. 2006. Acute andchronic vascular rejection in nonhuman primate kidney transplantation. Am
J Transplant. 6:1285–1296.

Received April 19, 2007
Accepted November 27, 2007
Corresponding Editor: Jill Pecon-Slattery