Download Differential Functional Variability of Serotonin Transporter and

Behavior Genetics ( 2005)
DOI: 10.1007/s10519-005-9017-8
Differential Functional Variability of Serotonin Transporter and
Monoamine Oxidase A Genes in Macaque Species Displaying
Contrasting Levels of Aggression-Related Behavior
Jens R. Wendland,1,6 Klaus-Peter Lesch,1 Timothy K. Newman,2 Angelika Timme,3 Hélène
Gachot-Neveu,4 Bernard Thierry,4 and Stephen J. Suomi5
Received 18 Jan. 2005—Final 17 June 2005
Functional allelic variation in the transcriptional control region of the serotonin transporter
and monoamine oxidase A genes has been associated with anxiety- and aggression-related
behavior in humans and, more recently, in nonhuman primates. Here, we have genotyped
these polymorphic regions in seven species of the genus Macaca. Macaques exhibit
exceptional inter-species variation in aggression-related social behavior as illustrated by
recent studies showing overlapping patterns of aggression-based social organization grades
and macaque phylogeny. We cloned and sequenced two new alleles of the serotonin
transporter gene-linked polymorphic region in Barbary and Tibetan macaques. In addition,
we observed that species displaying tolerant societies, with relaxed dominance and high
levels of conciliatory tendency, were monomorphic for both the serotonin transporter gene
and, with the exception of Tonkean macaques, the monoamine oxidase A gene. In contrast,
those species known to exhibit intolerant, hierarchical and nepotistic societies were
polymorphic at one or more of these loci. Rhesus (M. mulatta), the most intolerant and
hierarchical species of macaques, showed the greatest degree of allelic variation in both
genes. Additional investigation of a polymorphic repeat in exon III of the dopamine
receptor D4 as well as a repeat/single nucleotide polymorphism in the 3¢untranslated region
of the dopamine transporter which have both been implicated in the modulation of complex
behavior failed to reveal a relationship between allelic variability and social organization
grade. Taken together, these findings suggest that genetic variation of serotonergic
neurotransmission may play an important role in determining inter-species differences in
aggression related behavior in macaques.
KEY WORDS: Allelic variation; behavioral genetics; evolution; nonhuman primates.
1
2
3
4
Clinical and Molecular Psychobiology, Department of Psychiatry and Psychotherapy, University of Würzburg, 97080
Würzburg, Germany.
National Institute on Alcohol Abuse and Alcoholism, Laboratory of Clinical Studies-Primate Unit, Poolesville, MD 20837,
USA.
Institute of Human Biology and Anthropology, Freie Universität
Berlin, 14195 Berlin, Germany.
Centre d’Ecologie et Physiologie Energétiques, Centre National
pour la Recherche Scientifique, Strasbourg, France.
5
6
National Institute on Child Health and Human Development,
Laboratory of Comparative Ethology, Poolesville, MD 20837,
USA.
To whom correspondence should be addressed at Laboratory of
Clinical Science, National Institute of Mental Health, 10 Center
Dr.; Bldg. 10, Rm. 3D41, Bethesda, MD 20892-1264, USA. Tel.:
+1-301-496 3421; Fax: +1-301-402 0188; e-mail: wendlandj@
mail.nih.gov
2005 Springer Science+Business Media, Inc.
Wendland et al.
INTRODUCTION
In search of genetic variation influencing behavior, a
significant role for neurotransmission, as well as evidence of functionality, are considered to be crucial
(Ebstein et al., 2002). In humans, two genes of the
serotonin (5-hydroxytryptamine, 5-HT) pathway
meeting these criteria are the 5-HT transporter (5HTT, SLC6A4), which has been associated with
anxiety- and depression-related traits (Caspi et al.,
2003; Lesch et al., 1996); and monoamine oxidase A
(MAOA), which is believed to influence aggressive
behavior (Caspi et al., 2002; Deckert et al., 1999; see
Munafo et al., 2003 and Sen et al., 2004 for reviews).
Both the human 5-HTT and MAOA genes contain
gene-linked polymorphic regions (LPR) located upstream of the coding sequence, termed 5-HTTLPR
and MAOALPR, respectively. These polymorphisms
have been shown in vitro to alter the transcriptional
activity of 5-HTT and MAOA, and subsequently the
function of the corresponding proteins (Deckert
et al., 1999, Denney et al., 1999; Lesch, 2002, 1996;
Sabol et al., 1998).
In nonhuman primates, orthologous functional
polymorphic regions have been identified in rhesus
macaques (Macaca mulatta), designated as
rhMAOALPR and rh5-HTTLPR, respectively (Bennett et al., 2002; Lesch et al., 1997; Newman et al.,
2005). Several recent studies suggest that rh5HTTLPR genotypes interact with deleterious early
rearing experience in captive rhesus macaques to
influence orientation, attentional and affective capabilities (Champoux et al., 2002), sensitivity to ethanol
(Barr et al., 2003a, 2004a) and limbic-hypothalamicpituitary-adrenal axis stress response (Barr et al.,
2004b). With regard to aggressive behavior, a significant genotyperearing interaction in male rhesus
macaques has recently been demonstrated for the
rhMAOALPR (Newman et al., 2005).
Geographically one of the most widely distributed species of nonhuman primates, rhesus macaques
exhibit a degree of variation in aggression-related
behavior that is unequalled in other primate species
(Petit et al., 1997; Thierry, 1985; Thierry, 1990; de
Waal and Luttrell, 1989). Thierry (2000) recently
proposed a four-grade classification scheme for
describing social organization across 16 species of the
genus Macaca. This classification is based on the
extent and asymmetry of aggression-related behavior
within specific macaque species. Grade 1 species
exhibit highly hierarchical and nepotistic societies as
well as low levels of conciliatory behaviors, while
grade 4 species can be considered as more tolerant,
displaying relaxed dominance, open relationships and
high levels of conciliatory behaviors. For example,
the risk of a retaliatory attack from a subordinate is
much higher in grade 4 species than in grade 1 after
an initial attack by a dominant, but the retaliatory
attack will likely be much less severe, and the probability of reconciliation much higher, in grade 4 than
grade 1 macaques (Thierry, 2000). By mapping this
distribution of social style grades onto phylogenetic
trees provided by Purvis (1995) and Morales and
Melnick (1998), a significant association with phylogeny was shown for 7 of the 16 traits used for the
classification, including patterns of female rank
acquisition and male-female dominance relationships
(Thierry et al., 2000). This led to the conclusion that
the structure of macaque social organization is
influenced more by phylogeny than environment.
Thus, it is reasonable to hypothesize that specific
genetic polymorphisms contribute to the variability
in species-level behavior. In order to provide a
molecular basis for this hypothesis, we genotyped
rh5-HTTLPR and rhMAOALPR in seven macaque
species representing the spectrum of different social
grades. In addition, we genotyped (1) a physiologically relevant repeat polymorphism in exon III of the
dopamine receptor subtype 4 gene (DRD4) that has
been associated with novelty seeking in humans
(Benjamin et al., 1996; Ebstein et al., 1996), and (2) a
functional repetitive element/single nucleotide polymorphism in the 3¢untranslated region of the dopamine transporter gene (DAT 3¢-UTR) which has been
shown to be suggestive though not predictive of
hyperactive behavior in rhesus macaques (Miller
et al., 2001).
MATERIALS AND METHODS
Animals
Macaca fascicularis. A total of 35 longtail macaques (19f, 16m) originated from two independent
sources: (1) CIBA Vision, Strasbourg, France (12f,
13m, average pairwise relatedness <2%) and (2) the
AAP Sanctuary for Exotic Animals, Almere, The
Netherlands (7f, 4m, no data on kinship available).
Macaca nemestrina. Twelve pigtail macaques (7f,
5m) were included in this study, two of which were
unrelated females from the Centre de Primatologie,
Strasbourg. The remainder originated from the
above-mentioned AAP Sanctuary (no kinship data
available) in Almere.
Serotonin Transporter and MAOA Genes Variability in Macaques
Macaca tonkeana. A total of 28 Tonkean macaques (10f, 18m) from the Centre de Primatologie at
Strasbourg originated from four maternal lineages,
which in turn emerged from a stock of seven breeders
imported to France in 1972.
Macaca sylvanus. The sample population of 87
Barbary macaques (60f, 27m) analyzed in this study
was composed of four independent groups: (1) 13
males from the AAP Sanctuary in Almere; (2) 16
individuals (8f, 8m) from Naturzoo Rheine, Germany; (3) 46 females from Affenberg Salem, Germany; and (4) 12 individuals (6f, 6m, originally
imported from Gibraltar) from Wildpark Daun,
Germany. No data on kinship were available for
these groups.
Macaca thibetana and Macaca arctoides. Three
Tibetan (Macaca thibetana, one female and male each
that were unrelated and their son) and 2 unrelated
stumptail macaques (Macaca arctoides, 1f, 1m) originated from the Centre de Primatologie, Strasbourg.
Macaca mulatta. A total of 90 rhesus macaques
(28f, 62m, no kinship data available) originated from
the Deutsches Primatenzentrum, Göttingen, Germany (23f, 57m), and the AAP Sanctuary (5f, 5m).
Both groups were independent of each other. These
samples were genotyped only for the exon III polymorphism in the DRD4, as we and others have previously reported on rh5-HTTLPR (Bennett et al.,
2002; Lesch et al., 1997), rhMAOALPR (Newman
et al., 2005) and DAT 3¢-UTR (Miller et al., 2001).
Polymerase Chain Reaction Genotyping and Data
Analysis
All protocols were approved by the relevant local animal care and use committees. After i.m. anesthetization with 15 mg/kg ketamine, peripheral blood
was drawn and DNA extracted according to standard
procedures. 5-HTTLPR was amplified using oligonucleotide primers rhMUT 5¢-TCG ACT GGC GTT
GCC GCT CTG AAT GC-3¢and rhINT 5¢-CAG
GGG AGA TCC TGG GAG GGA-3¢under reaction
conditions previously described (Lesch et al., 1997).
MAOALPR was amplified using primers MAOAjrwF2 5¢-AGA AGG GCT GCG GGA AGC-3¢ and
MAOA-jrwR 5¢-GTG CTC CAC TGG GAA CTG
G-3¢under the following reaction conditions; 25–
50 ng DNA were mixed with reaction buffer containing 1.8 mM MgCl2, 4% DMSO, 0.2 mM dNTP
each, 400 nM forward and reverse primer each and
1.25 U of Roche FastStart HighFidelity Taq polymerase in a final reaction volume of 25lL; amplifi-
cation conditions were 2 min initial denaturaion at
95C followed by 35 cycles of 95¢C–65C–72C (30 s
each) and a final elongation for 5 min at 72C.
Amplification and restriction endonuclease digestion
conditions followed the published protocols of
Ebstein et al. (1996) for the DRD4 exon III polymorphism, and Miller et al. (2001) for DAT 3¢-UTR.
Amplicons were separated by 3.5% NuSieve agarose
gel electrophoresis and visualized by ethidium bromide (Sigma) staining. Genotypes were assigned
based on estimated allele sizes established using
sequenced controls from prior experiments. In the
case of 5-HTTLPR, two new alleles were identified,
cloned with the TA Cloning Dual Promoter Kit
(Invitrogen) and sequenced by automated cycle
sequencing employing the fluorescent dye termination
method (ABI-Prism 310). Sequence alignment for
5-HTTLPR in Figure 1 was performed using human
S, L, xL and xxL alleles (GenBank accession numbers
X76753 and Y13147) and rhesus rhS, rhL and rhxL
alleles (AF191557).
In every species analyzed, observed genotype
frequencies did not significantly deviate from Hardy–
Weinberg equilibrium as determined by v2 test. Also,
all allelic frequencies presented in this study did not
significantly differ between sexes.
RESULTS
Serotonin Transporter Gene-linked Polymorphic
Region
We have previously reported on three different
rh5-HTTLPR alleles in rhesus macaques, namely rhS,
rhL and rhxL (Bennett et al., 2002; Lesch et al.,
1997). In this study, we identified two additional
alleles: one in Barbary macaques, which we designate
msy (for Macaca sylvanus, GenBank accession number AY897212); the other one in Tibetan macaques,
designated mti (AY897213). Sequence alignment
revealed that the mti allele, which is 23 bp shorter
than rhS, has most likely evolved from a deletion of
three repetitive motifs in the second polymorphic locus (PL2) described previously (Fig. 1) (Lesch
et al., 1997). The msy allele has almost exactly the
same length as rhxL, but it displays an additional
repetitive unit in PL2 and lacks the insertion further
downstream of the rhxL allele (PL3, Fig. 1). All of
the other macaque species investigated carried the
rhL allele. In addition, all species analyzed, except for
rhesus macaques, were monomorphic at this locus
(Table I).
Wendland et al.
Fig. 1. Multiple sequence alignment of 5-HTTLPR alleles in humans and macaques. For alignment optimization and comprehensibility,
hyphens were introduced and repetitive units separated by spaces. S, L, xl and xxL refers to human alleles described previously (GenBank acc.
nos. X76753, Y13147); rhS, rhL and rhxL alleles were first observed in rhesus macaques (AF191557). Mti, Tibetan macaque allele
(AY897213); msy, Barbary macaque allele (AY897212); PL, polymorphic locus. Note that variation in humans (PL1) and macaques (PL2 and
3) occurs at different, albeit close, genetic loci.
Monoamine Oxidase A-linked Polymorphic Region
Alleles in the promoter region of the MAOA
gene had 5–7 repeats of the 18 bp repetitive sequence,
designated rh5, rh6 and rh7, as described previously
for rhesus macaques (Newman et al., 2005; Wendland et al., in press). Barbary macaques were monomorphic, carrying the rh6 allele, whereas stumptail
and Tibetan macaques were monomorphic for the
rh7 allele (Table I). The remaining macaque species
were polymorphic: pigtail (rh6 and rh7), Tonkean
(rh6 and rh7), longtail (rh6 and rh7) and rhesus
macaques (rh5, rh6 and rh7, Table I). Thus, as with
the 5-HTTLPR locus, variability of MAOALPR alleles was greatest in rhesus macaques.
Dopamine Receptor D4 Exon III Polymorphism
We observed only two DRD4 exon III alleles
containing either 4 or 5 repeats. All Barbary
macaques were monomorphic for the 4 repeat allele,
while all Tibetan, pigtail and stumptail macaques
were monomorphic for the 5 repeat allele. Rhesus,
longtail and Tonkean macaques displayed polymorphism at the DRD4 locus with 4 and 5 repeat
alleles, the frequencies of which are shown in
Table I.
Dopamine Transporter 3¢Untranslated Region
Polymorphism
All samples analyzed were monomorphic,
exhibiting only the 12 repeat allele, with the exception
of Tonkean and pigtail macaques (11 and 12 repeat
alleles, Table I). The single nucleotide polymorphism
(SNP) in the last repetitive unit recognized by the
restriction enzyme Bst1107I varied only in pigtail
macaques and in two Barbary macaques, both of
which were heterozygous (Table I).
SNP+
SNP)
rh6 (47%)
rh7 (53%)
rh6 (26%)
rh7 (74%)
SNP+ (100%)
4 (19%)
5 (81%)
12 (100%)
rhL (100%)
rhL (100%)
rhS (26%)
rhL (74%)
rhxL (0.2%)
rh5 (35%)
rh6 (25%)
rh7 (40%)
4 (89%)
5 (11%)
12 (100%)
11 (8%)
12 (92%)
SNP+ (63%)
SNP) (37%)
5 (100%)
12 (5/7)
35 (16/19)
22–289b,c,d
2
M. nemestrina
(Pigtail m.)
2
M. fascicularis
(Longtail m.)
1
M. mulatta
(Rhesus m.)
SNP+ (99%)
SNP) (1%)
12 (100%)
4 (100%)
rh6 (100%)
msy (100%)
87 (27/60)
3
M. sylvanus
(Barbary m.)
SNP+
12
5
rh7
mti
3 (2/1)
3
M. thibetana
(Tibetan m.)
SNP+
12
5
rh7
rhL
2 (1/1)
3
M. arctoides
(Stumptail m.)
4 (25%)
5 (75%)
11 (18%)
12 (82%)
SNP+ (100%)
rh6 (34%)
rh7 (66%)
rhL (100%)
28 (18/10)
4
M. tonkeana
(Tonkean m.)
Seven different macaque species of all four social style grades as proposed by Thierry (2000) were genotyped. With the exception of Macaca tonkeana, macaque species exhibiting more
tolerant societies (i.e. grades 3 and 4) carry only one kind of allele for 5-HTTLPR and MAOALPR each. In contrast, intolerant, hierarchical and nepotistic macaque species (grades 1
and 2) are polymorphic at one or more of these loci.
a
SNP +/) refers to the Bst1107I restriction enzyme polymorphism in the last repetitive unit of DAT 3¢-UTR.
b
Data for rh5-HTTLPR (n=289) were pooled from previous experiments (Bennett et al., 2002; Lesch et al., 1997).
c
rhMAOALPR data (n=217) are taken from Newman et al. (2005).
d
DAT 3¢-UTR data (n=22) from Miller et al. (2001); for DRD4 in rhesus macaques, n=90. Allelic frequencies were omitted for Tibetan and stumptail macaques due to small sample
size and for DAT 3¢-UTR SNP in rhesus macaques due to missing details in cited work.
DAT 3¢-UTRa
DRD4 exon III
MAOALPR
Social organization category
sensu Thierry
Number of animals
(male/female)
5-HTTLPR
Macaque species
Table I. Distribution of Alleles and Respective Frequencies in Polymorphic Genetic Loci Presumably Involved in Behavior
Serotonin Transporter and MAOA Genes Variability in Macaques
Wendland et al.
DISCUSSION
The present study has analyzed seven species of
the genus Macaca for functional polymorphisms in
genes of the serotonergic and dopaminergic pathway
that are assumed to influence behavior. We have
shown that genetic loci involved in the regulation of
serotonergic neurotransmission, especially the
5-HTTLPR, vary not only in their sequence composition, but also in their allelic frequencies across
macaque species. The presented data provide a
molecular basis for future association studies of
individual behavior with functional genetic variants
in a novel inter-species approach within one genus of
nonhuman primates.
Macaques were investigated because of the
behavioral classification developed by Thierry (2000)
that is based on inter-species differences in aggression-related behavior, and also because of recent
evidence for the involvement of rh5-HTTLPR
(Champoux et al., 2002) and rhMAOALPR
(Newman et al., 2005) in aggressive behavior, affective capacities, and emotion regulation in rhesus
macaques. Moreover, it has been shown that Thierry’s social style grades cluster within species groups
in a manner that is consistent with their phylogenetic
relationships (Thierry et al., 2000). Our data show
that macaque species (except Tonkean) exhibiting less
aggressive, more tolerant behavior (grades 3 and 4)
displayed no intraspecific variation in 5-HTTLPR
and MAOA-LPR genotypes. In contrast, highly
intolerant and hierarchical macaque species (grades 1
and 2) were polymorphic at one or both of these loci
(Table I).
The genetic loci analyzed in this study have been
chosen, on the one hand, on the evidence of association with human personality traits as well as the
presence of geneenvironment effects in captive
rhesus macaques and, on the other hand, due to
in vitro data showing an effect upon the molecular
physiology of neurotransmission. The first gene variant to be associated with a human personality trait,
in this case novelty seeking, was a polymorphism in
the third cytoplasmatic loop of DRD4 (Benjamin
et al., 1996; Ebstein et al., 1996). This polymorphism
consists of 2–10 repetitive units of 48 bp each and is
believed to influence second messenger signaling
(Asghari et al., 1995; Kazmi et al., 2000; Lichter
et al., 1993;Van Tol et al., 1992). Alleles with 4 or 7
repeats account for the vast majority of all alleles in
humans (Lichter et al., 1993; Van Tol et al., 1992). In
our study, macaques only carried alleles of the same
size as human 4 and 5 repeat variants, and there was
no apparent relationship between the degree of variability in this polymorphic locus and the social grade
of macaque species (Table I).
The second functional polymorphism in the
dopaminergic pathway analyzed in this study was
DAT 3¢-UTR. This variable number of tandem repeat
(VNTR) polymorphism comprises 3–11 copies of a
40 bp motif in humans and has, among others, been
associated with attention deficit hyperactivity disorder (Cook et al., 1995; Gill et al., 1997; Sano et al.,
1993; Vandenbergh et al., 1992). Possibly as a cisacting regulatory determinant of mRNA half-life,
this VNTR polymorphism has been shown in vitro to
alter reporter gene expression (Conne et al., 2000;
Fuke et al., 2001). Only one 12-repeat allele is known,
to date, to exist in rhesus macaques, but a SNP in the
last repetitive unit of VNTR that is recognized by the
restriction enzyme Bst1107I was suggestive (though
not predictive) of hyperactive behavior in a sample of
22 adult male rhesus macaques (Miller et al., 2001).
In longtail macaques, alleles with 11 and 12 repeats
have recently been described, exhibiting significantly
different activities on reporter gene assays (InoueMurayama et al., 2002). We observed an allele of 11
repeats in Tonkean (allelic frequency 18%) and pigtail macaques (in 2 out of 24 alleles), but not in
longtail macaques (Table I). Regarding the Bst1107I
polymorphism, only Barbary and pigtail macaques
were polymorphic, while all other analyzed species
lacked the restriction site. As in the case of DRD4, we
found no relationship between macaque social style
grade and within-species variability of the DAT
3¢-UTR VNTR or SNP.
In serotonergic neurotransmission, the 5-HTT
plays a key role in controlling intrasynaptic availability of 5-HT (reviewed in Murphy et al., 2004).
The human 5-HTT was the second gene for which a
positive association with personality traits was shown
(Lesch et al., 1996). The variation consists of a 43 bp
insertion/deletion in the transcriptional control region which affects reporter gene expression in vitro
(Lesch et al., 1996). In numerous follow-up studies,
the initial observation of association with anxietyand depression-related traits was replicated in several
but not all studies (for review see, e.g., Lesch, 2002;
Sen et al., 2004). Recent data from a prospectivelongitudinal birth cohort study (Caspi et al., 2003)
have shown that individuals carrying one or two
copies of the S allele are more likely to exhibit
diagnosable depression after stressful life events than
L/L genotype individuals, supporting a role as
Serotonin Transporter and MAOA Genes Variability in Macaques
susceptibility gene. To date, at least 14 different alleles have been discovered in humans, 6 in great apes
(Hominidae), 5 in Müller’s gibbons (Hylobates muelleri) and 3 in rhesus macaques (Bennett et al., 2002;
Inoue-Murayama et al., 2000; Lesch et al., 1997;
Nakamura et al. 2000). While the functional implications for most of these alleles remain unclear, a
significant difference in reporter gene expression was
demonstrated for the human S and L alleles as well as
rhesus S and L alleles (Bennett et al., 2002; Lesch
et al., 1996). In humans this functional dichotomy
was subsequently confirmed by different approaches
at both the pre- and postsynaptic level (David et al.
2005, reviewed in Lesch, 2002). Here, we observed
two novel alleles in Barbary and Tibetan macaques,
both of which displayed sequence differences from
rhS and rhL in the PL2 described previously (Fig. 1,
Lesch et al., 1997). Interestingly, the msy allele carries the highest number of repeats in PL2 of all macaques. Bearing in mind that macaques represent a
monophyletic lineage (Delson, 1980) and that Barbary macaques were the first species to diverge from
this clade (Morales and Melnick, 1998), the msy allele
thus comes closest of all currently available sequences
to the ‘‘progenitor’’ 5-HTTLPR allele postulated
previously (Lesch et al., 1997). Moreover, our data
suggest that, with the exception of the very rare rhxL
allele, macaque 5-HTTLPR alleles could have
evolved from an msy-like ancestral allele by deletion
of repetitive units in the PL2 (Fig. 1).
The usefulness of the nonhuman primate model
in the study of behavioral genetics is generally
attributed to the ability to control and manipulate the
animals’ environment (Barr et al., 2003b). Here,
however, we have used a complementary approach by
comparing species within the genus Macaca displaying different patterns of aggression-based social
organization. The findings presented in this study
support the importance of the central serotonergic
system in behavioral genetics. Numerous association
studies have looked at correlations between genetic
polymorphisms and traits in considerably diverse
species including, in particular, humans, nonhuman
primates and rodents. By contrast, our study represents a novel attempt towards elucidating the genetic
basis of social behavior by comparing well-established
functional polymorphisms in genes of the serotonergic or dopaminergic pathway in different species
within one genus. Macaque species are ideally suited
to this purpose since they exhibit broad interspecific
variation in patterns of aggression, dominance and
temperament (Aureli et al., 1997; Petit et al., 1997;
Thierry, 1985; Thierry, 1990; Thierry, 2000; de Waal
and Luttrell, 1989). Moreover, it was shown for this
genus that social organization and behavior have
changed little during several hundred thousand years
(Thierry et al., 2000). Ecological and climatic conditions are likely to have varied considerably during
such a long period of time, and if one assumed that
macaque behavior and social organization were solely influenced by environment, one should also expect changes in behavior as an expression of
adjustment to changing peristasis. Instead, however,
the influence of phylogeny was proposed by several
studies (Chan, 1996; Matsumura, 1999; Thierry,
2000). In line with this hypothesis, Thierry and
coworkers demonstrated significant association of his
classification of social aggression in macaques based
on two independently derived phylogenies (Thierry
et al., 2000). In a similar approach investigating rodents of the genus Microtus known to display strong
species differences in social structure, a significant
effect of a polymorphic microsatellite upstream of the
avpr1a gene upon in vitro and in vivo gene expression
and social engagement and bonding behavior was
recently demonstrated (Hammock and Young, 2005).
Our findings reveal that functional polymorphisms
in the serotonergic pathway vary both within and between macaque species: tolerant, less hierarchical and
nepotistic macaque species (grades 3 and 4) were
monomorphic at rh5-HTTLPR and rhMAOALPR
(Barbary, Tibetan and stumptail macaques) or displayed only two alleles for rh5-HTTLPR (Tonkean
macaques). In contrast, highly asymmetrically aggressive and hierarchical species (grades 1 and 2) were
polymorphic in the serotonergic pathway, and the most
intolerant species (rhesus macaques, grade 1) also displayed the largest number of alleles at both loci. In
summary, we feel it is justified at this point to
hypothesize that rh5-HTTLPR and rhMAOALPR
gene variants affect key elements of macaque social
behavior, in particular the exceptional level of interspecies variation in aggression-related behavior in
macaques.
Certainly, despite its intrigue, our conclusion
bears several caveats. Firstly, although all individuals
in each species except for Tibetan and stumptail
macaques originated from at least two independent
sources (4 sources in the case of monomorphic Barbary macaques), we cannot rule out the possibility of
having missed alleles through founder effects in the
colonies from which our samples were derived. Secondly, the relative paucity of rh5-HTTLPR and
rhMAOALPR variation in grade 3 and 4 species
Wendland et al.
could be due to statistical chance; for example, it has
been calculated that one would need a minimum
sample size of n=51 to detect all alleles with frequencies of ‡10% at the 95% confidence level
(Gregorius, 1980). Finally, it would clearly not be
justified to deduce social or behavioral variability
from genetic variability at the population level as
long as data on both behavior and genotype on
individual subjects are lacking. Nevertheless, our
results provide a first molecular basis to create falsifiable hypotheses for the study of specific aspects of
social behavior, such as aggression, in a behaviorally
exceptionally diverse and well-characterized genus of
non-human primates.
Therefore, to corroborate or disprove our
hypothesis of an involvement of rh5-HTTLPR and
rhMAOALPR underlying variability in social
aggression, analyses of larger sample populations
derived from unrelated individuals, as well as investigations of more macaque species of all grades are
needed. Ideally, one would have behavioral and
genotype data on individuals representing macaques
of all species to perform association studies. Owing to
5HT system’s tremendous complexity with more than
a dozen receptors and serotonergic nerve endings
widely distributed in the CNS, we need an integrative
model for serotoninergic neurotransmission spanning
from seemingly simple molecular variations such as
5-HTTLPR and MAOALPR and synapse homeostasis to a highly complex phenomenon such as interindividual behavior in order to better understand the
way in which genes might affect primate behavioral
traits.
DISCLAIMER
In compliance with NIH/NIMH ethics guidelines, we hereby state that the views expressed do not
necessarily represent those of NIH or NIMH or the
federal government of the USA.
ACKNOWLEDGMENTS
The authors thank Gabriela Ortega, Nicole
Steigerwald and Mukta Kundu for excellent technical
assistance in blood and DNA sample processing,
genotyping, cloning and data collection. We are
greatly indebted to G. Flügge, Deutsches Primatenzentrum, Göttingen, Germany; I. den Hartog and
E. van Lavieren, AAP Sanctuary for Exotic Animals,
Almere, The Netherlands; A. Johann, Naturzoo
Rheine, Germany; G. de Tuerkheim, E. Merz and
W. Angst, Affenberg Salem, Germany; S. Bost and
D. Reichle, Wildpark Daun, Germany; and CIBA
Vision, Strasbourg, France, for providing blood
samples of different macaque species. Supported by
the Division of Intramural Research, National
Institute of Child Health & Human Development,
National Institutes of Health and the European
Commission
(NEWMOOD
LSHM-CT-2003–
503474) and the Deutsche Forschungsgemeinschaft
(SFB 581, KFO 125/1–1).
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Edited by Jeanne Wehner