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NEWS AND PERSPECTIVES
Heritability of digit ratio (2D:4D) in rhesus macaques
(Macaca mulatta)
Emma Nelson •Martin Voracek
Received: 3 September 2009 / Accepted: 1 October 2009
ÓJapan Monkey Centre and Springer 2009
Abstract The second-to-fourth digit ratio (2D:4D) is a
putative biomarker for prenatal androgen effects, which has
been widely employed to study androgenic-programming
effects on shaping sex-linked traits and behaviours in
humans. This approach is now increasingly applied to non-
human species. Heritability studies of 2D:4D in both humans
and zebra finches indicate substantial genetic contributions
to the expression of this trait. This study examines the her-
itability of 2D:4D in rhesus macaques, based on the resem-
blance of mother–infant dyads, to see how these compare
with human values. Results suggest that familial resem-
blance in 2D:4D is also strong in rhesus monkeys. Herita-
bility estimates were within the range of estimates from
human studies. These preliminary results suggest that the
strength of heritability of 2D:4D may generalize across taxa.
Keywords Digit ratio (2D:4D) Prenatal testosterone
Sex differences Mother–offspring resemblance
Rhesus macaques
Introduction
In humans the second-to-fourth digit ratio (2D:4D) is a
putative biomarker for prenatal androgen effects (Manning
2002). Although there is substantial overlap between the
sexes, 2D:4D tends to be lower in males than females, and
sex differences are quite stable through growth (Manning
et al. 2004). Although the aetiology of the sex effect in
2D:4D is not yet clear, it is commonly believed to be un-
derpinned by HOX genes shared between the distal limb
buds (digits) and the genital bud (Manning 2002;Za
´ka
´ny
et al. 1997).
2D:4D has been linked to various conditions associated
with the prenatal androgen milieu. For example, a high
testosterone-to-estradiol ratio in the amniotic fluid is
associated with lower (masculinized) 2D:4D in children
(Lutchmaya et al. 2004); women with congenital adrenal
hyperplasia (i.e., who were exposed to high prenatal
androgen levels) have masculinized 2D:4D (O
¨kten et al.
2002); and low 2D:4D in men is associated with higher
genetically-based sensitivity to testosterone (Manning et al.
2003). These, and other convergent lines of evidence, have
led to 2D:4D being widely employed to study androgenic-
programming effects on shaping human sex-linked traits
and behaviours (Voracek and Loibl 2009). The biomarker
is now increasingly implemented in animal studies (Lom-
bardo and Thorpe 2008).
One line of 2D:4D research is concerned with teasing
apart genetic and non-genetic influences on digit ratios in
an attempt to understand how 2D:4D is inherited across
generations. This is quantified by calculating heritability
(h
2
) estimates for the trait from genetically informative
studies. For humans, twin studies present the best non-
molecular model for investigating the genetic inheritance
of traits. Therein, heritabilities are calculated from the
differential resemblance of identical (monozygotic) twins
(genetically identical and experiencing the same environ-
ment) versus fraternal (dizygotic) twins (also experiencing
the same environment, but, like siblings, sharing only 50%
E. Nelson (&)
School of Archaeology, Classics and Egyptology,
University of Liverpool, Hartley Building,
Brownlow Street, Liverpool L69 3GS, UK
e-mail: enelson@liverpool.ac.uk
M. Voracek
Department of Basic Psychological Research,
School of Psychology, University of Vienna,
Vienna, Austria
123
Primates (2010) 51:1–5
DOI 10.1007/s10329-009-0173-y
of their genes). This way, genetic factors can be separated
from environmental influences on the trait.
In contrast, studies based on familial relationships (i.e.,
parent–offspring or sibling–sibling dyads) cannot disen-
tangle these two sources. However, four human twin
studies of 2D:4D (Paul et al. 2006; Voracek and Dressler
2007; Gobrogge et al. 2008; Medland and Loehlin 2008)
have concordantly found high heritabilities for the trait
(h
2
=50–80%) and shared postnatal environmental influ-
ences on 2D:4D to be negligible or nil. Hence, family
studies can be used to estimate the heritability of 2D:4D
because, through the lack of influences of the shared
environment, the family-based heritability estimates will
not be inflated (see Marshall 2000; Manning et al. 2001;
Voracek and Dressler 2009). Of note, two further reports
(Forstmeier 2005; Forstmeier et al. 2008) have also indi-
cated high heritability (h
2
=70–80%) of 2D:4D in zebra
finches.
To understand whether heritability levels of 2D:4D
generalise across taxa, a comparative framework is needed.
Given that the main body of 2D:4D research is in humans,
a framework considering species closely related to humans
would be the most informative. Haplorrhine primates
undoubtedly offer the best model, as they reside within the
same suborder and will therefore have more similar bio-
logical profiles to humans than more distantly related taxa,
such as birds or reptiles. Hence, the aims of this small case
study were primarily to use a family-resemblance approach
to estimate the heritability of 2D:4D in mother–offspring
dyads of rhesus macaques (Macaca mulatta) to see how
this compares with human evidence and, secondarily, to
provoke further research in this field.
Methods
Study population and study group
Data were collected during trapping season (early in 2008)
on Cayo Santiago, a small island (15.2 ha) located 1 km
off Puerto Rico’s south-eastern coast. The free-ranging
rhesus macaque colony is managed by the Caribbean Pri-
mate Research Center (CPRC) and was established
70 years ago. The present-day population (about 900
individuals) is directly descended from the founder group,
originating from the Indian subcontinent (Rawlings and
Kessler 1986). Genetic and behavioural analyses indicate
no significant inbreeding effects (McMillan and Duggleby
1981; Mason and Perry 1993; Charpentier et al. 2007),
although female dispersal patterns may potentially reduce
genetic diversity (Chepko-Sade and Sade 1979). The study
sample was obtained within a larger, long-term study
investigating ageing in rhesus monkeys (unrelated to this
study), for which animals had to be anaesthetised. Our
sample size was therefore restricted to those individuals
being sedated for the larger study. Sampled were 25 mature
females and their 25 infants (eight females, 17 males).
Adult females were aged between 4 and 23 years (birth
dates were known to the day), and infants (all from the
same birth season, 2007/2008) between 1 and 4 months.
Measuring procedure
Monkeys were trapped between 08:30 and 12:00 inside
feeding corrals, where they receive food throughout the
year. The trained staff of the CPRC captured the monkeys
in nets or by hand and immediately transferred them to
cages. Females and their infants were held in the same
cages overnight. Individuals had access to water and
monkey chow during this period. The following morning,
females and their infants were kept together and anaes-
thetised with ketamine (approximately 10 mg/kg IM
injection). About 10 min after ketamine injection, mor-
phometric measures were taken. Data were collected from
25 mother–infant pairs. While the individuals were fully
immobilised, the lengths of the second and fourth digits
of each hand were measured to the nearest 0.01 mm,
using a digital vernier calliper, from the crease at the base
of the digit proximal to the palm to the tip of the
extended digit (Manning 2002). Then, the same observer
repeated the digit measurements. Subsequently, trained
veterinary staff monitored the monkeys every 10 min
until they fully recovered from the procedure. Once fully
alert, monkeys were offered water and monkey chow and
were then released back into their social groups. All
animals made uneventful recoveries and behaved nor-
mally afterwards.
Data analysis
Right-hand and left-hand digit ratio (R2D:4D and L2D:4D)
was calculated by dividing the mean of the first and second
measurements of the second digit (2D) by the mean of the
first and second measurements of the fourth digit (4D).
Intraobserver measurement repeatabilities were quantified
with intraclass correlation coefficients (ICC), according to
a two-way mixed-effects model with absolute-agreement
definition (Voracek et al. 2007b). The ICC values were
0.69 and 0.65 for mother and infant R2D:4D, and 0.91 and
0.78 for mother and infant L2D:4D (all Ps\0.02). Inde-
pendent groups and paired ttests were used for group and
right-left comparisons of 2D:4D. Following standard
practice of family studies (Voracek and Dressler 2009),
heritability estimates were obtained by doubling the Pear-
son correlation coefficients (r) quantifying the mother–
offspring resemblance.
2 Primates (2010) 51:1–5
123
Results
Between-group comparisons of 2D:4D
Male infants had lower R2D:4D than female infants [mean
and standard deviation (SD): 0.808 and ±0.028 vs. 0.816
and ±0.034], although this difference was not nominally
significant with the available sample size (t=0.58,
df =23, two-tailed P=0.57, Cohen d=-0.27). Similar
results were obtained for L2D:4D (mean and SD of male vs.
female infants: 0.820 and ±0.025 vs. 0.833 and ±0.037;
t=1.09, df =23, P=0.29, d=-0.45). Infants’ R2D:4D
(mean and SD: 0.811 and ±0.030) and L2D:4D (0.824 and
±0.029) were weakly positively related (r=0.30,
P=0.15), and infants’ R2D:4D was somewhat, but not
significantly, lower than their L2D:4D (paired ttest:
t=1.91, df =24, P=0.07). Mothers’ R2D:4D (mean and
SD: 0.825 and ±0.026) and L2D:4D (0.822 and ±0.045)
were significantly positively related (r=0.50, P=0.01),
and mothers’ R2D:4D was commensurate with their
L2D:4D (paired ttest: t=-0.40, df =24, P=0.69).
Mothers’ R2D:4D significantly increased with age
(r=0.41, P=0.02) but not their L2D:4D (r=0.20,
P=0.34). Infants’ digit ratios exhibited no age effects.
Heritability of 2D:4D
With both sexes of infants combined for analysis, mother–
infant resemblance (based on 25 dyads) was strong and
significant for R2D:4D (r=0.58, P=0.002), whilst
somewhat less strongly and not nominally significant for
L2D:4D (r=0.31, P=0.13). Hence, the heritability
estimate was h
2
=62% for L2D:4D, whilst off the upper
boundary (i.e. h
2
[100%) for R2D:4D (Fig. 1).
With one exception (see below), these findings generally
held true in separate analyses, accounting for offspring sex.
Mother–son resemblance (based on 17 dyads) for R2D:4D
was r=0.62 (P=0.008) and r=0.42 (P=0.09) for
L2D:4D, whereas mother–daughter resemblance (based on
eight dyads), in above order, was r=0.53 (P=0.18) and
r=-0.12 (P=0.78), respectively.
Discussion
This is the first study to compare 2D:4D in infant rhesus
monkeys and to estimate the heritability of 2D:4D in a non-
human primate. Heritability estimates, based on this small
sample of mother–infant dyads, were found to be within the
range of human twin and family studies of 2D:4D. This
suggests that genetic contributions to the expression of
2D:4D generally are substantial and that this pattern might
generalise across higher taxonomic groups.
Among infant monkeys, males had slightly lower 2D:4D
than females. Thus, sex differences were in the expected
direction, representing small-to-medium-sized effects
(ds=-0.27 and -0.45). Among adult females, R2D:4D
increased with age. Similar age effects have been found for
adult female chimpanzees (McIntyre et al. 2009), whereas
in humans, 2D:4D increases slightly during childhood
(McIntyre et al. 2005) but appears to remain stable in
adulthood (Manning 2002). Age effects were not observed
in infant monkeys, which might be attributable to this
group’s narrow age range.
Heritability of 2D:4D in rhesus monkeys was significant
for mother–son dyads, but with a limited sample size, not
for mother–daughter dyads. Heritability was higher in the
right hand compared with the left. These results accord
with a recent large study of familial resemblance of 2D:4D
in humans (Voracek and Dressler 2009). Thus, genetic
effects appear to have less influence on L2D:4D, which
might suggest that environmental factors may be exerting
stronger influences on the left hand. For example, it has
been shown that human males and females infected with
Toxoplasma gondii (a protozoan parasite) have low
L2D:4D compared with the R2D:4D (Flegr et al. 2008).
This study design ensured that heritability estimates
were derived from mothers with very young infants, thus
minimising postnatal environmental influences on 2D:4D.
Conversely, it might have allowed heritability to be exag-
gerated by the exclusion of possible non-shared environ-
mental effects on 2D:4D occurring later in life, such as
age-related changes in adulthood. Additionally, and
importantly, the small sample size of this study makes
these results preliminary, although it is predicted that the
Fig. 1 Relationships between mother and infant digit ratios. Right-
hand second-to-fourth digit ratio (2D:4D) has higher heritability
(h
2
[100%) than left-hand 2D:4D (h
2
=62%)
Primates (2010) 51:1–5 3
123
h
2
estimates quoted here, particularly the slightly negative
correlation for L2D:4D (based on only eight mother–
daughter dyads) and the high h
2
estimates for R2D:4D in
general (which were off the upper boundary) conceivably
should stabilise in larger samples.
Because of the conformity of h
2
estimates for 2D:4D
from several human twin and family studies, two parent–
offspring studies of zebra finches, and the current mother–
offspring study of rhesus macaques, it may be conjectured
that high heritability levels of 2D:4D generalise across
higher taxonomic groups. A possible test of this hypothesis
would be a twin study of captive callitrichids, the only
family of primates consistently producing twins (Goldizen
2003). However, any study comparing across species must
control for phylogenetic effects (Felsenstein 1985).
Although these results are preliminary and with a small
sample size they allude to the possibility that 2D:4D may
be as useful to behavioural research in non-human primates
as it has been in human studies (see Voracek and Loibl
2009). We therefore urge researchers to extend this
research trajectory. Future studies of heritability should
aim for much larger samples, which include multigenera-
tional pedigree relationships (see Kruuk 2004), and, when
possible, address influences of assortative mating (see
Voracek et al. 2007a), consanguinity, founder effects (see
Chepko-Sade and Sade 1979) and other potentially relevant
developmental factors.
Acknowledgments Thanks are extended to Melissa Gerald, Christy
Hoffman, and the Caribbean Primate Research Center staff. All
procedures in this study adhered to US laws governing animal
research and were therefore in accordance with the National Institutes
of Health Guide for the Care and Use of Laboratory Animals. The
study protocol was approved by the Institutional Animal Care and
Use Committee of the University of Puerto Rico (PR, USA) and the
School of Archaeology, Classics and Egyptology’s Research Com-
mittee (University of Liverpool, UK).This publication complied with
animal care regulations and national laws and was made possible by
Grant P40RR003640 from the National Center for Research
Resources (NCRR), a component of the National Institutes of Health
(NIH). Its contents are solely the authors’ responsibility and do not
necessarily represent official views of NCRR or NIH. Funding sup-
port was provided by the University of Liverpool, the British Acad-
emy Centenary Project Fund and the British Association of Biological
Anthropology and Osteoarchaeology.
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