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INTERNATIONAL FEDERATION OF PIGMENT CELL SOCIETIES · SOCIETY FOR MELANOMA RESEARCH

PIGMENT CELL & MELANOMA

Research
The evolution of human skin pigmentation
involved the interactions of genetic,
environmental, and cultural variables
Nina G. Jablonski

DOI: 10.1111/pcmr.12976
Volume 34, Issue 4, Pages 707–729

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Received: 25 February 2021 | Revised: 30 March 2021 | Accepted: 3 April 2021

DOI: 10.1111/pcmr.12976

REVIEW

The evolution of human skin pigmentation involved the

interactions of genetic, environmental, and cultural variables

Nina G. Jablonski

Department of Anthropology, The

Pennsylvania State University, University
Park, PA, USA
Correspondence
Nina G. Jablonski, Department of
Anthropology, 409 Carpenter Building, The
Pennsylvania State University, University
Park, PA 16802, USA.
Email: ngj2@psu.edu
Abstract
The primary biological role of human skin pigmentation is as a mediator of penetra-
tion of ultraviolet radiation (UVR) into the deep layers of skin and the cutaneous
circulation. Since the origin of Homo sapiens, dark, protective constitutive pigmenta-
tion and strong tanning abilities have been favored under conditions of high UVR and
represent the baseline condition for modern humans. The evolution of partly depig-
mented skin and variable tanning abilities has occurred multiple times in prehistory,
as populations have dispersed into environments with lower and more seasonal UVR
regimes, with unique complements of genes and cultural practices. The evolution of
extremes of dark pigmentation and depigmentation has been rare and occurred only
under conditions of extremely high or low environmental UVR, promoted by positive
selection on variant pigmentation genes followed by limited gene flow. Over time,
the evolution of human skin pigmentation has been influenced by the nature and
course of human dispersals and modifications of cultural practices, which have modi-
fied the nature and actions of skin pigmentation genes. Throughout most of prehis-
tory and history, the evolution of human skin pigmentation has been a contingent and
non-­deterministic process.

KEYWORDS

bottleneck, depigmentation, dispersal, eumelanin, folate, tanning, ultraviolet radiation, UVB,

vasodilation, vitamin D

1 | I NTRO D U C TI O N was a breakthrough in evolution that contributed to the ability of

The evolution of mostly naked, darkly pigmented skin in the genus
Homo is often not accorded the same importance in human prehis-
tory as the evolution of a relatively large brain, a musculoskeletal
system adapted for sustained, striding bipedal locomotion, and the
manufacture and use of stone tools. This is mostly because, until re-
cently, details of the evolution of the human integument were ignored
or downplayed in the absence of direct fossil evidence. We now un-
derstand that the evolution of a skin interface, which facilitated sus-
tained high levels of physical activity in a sunny and hot environment,
hominins to occupy, adapt to, and modify diverse terrestrial environ-
ments. Significant expansion of the knowledge bases in paleontology,
archeology, genetics, genomics, and environmental physiology in re-
cent decades has made possible a new focus on the central role of
human skin and skin pigmentation in maintaining homeostasis and in
ensuring individual survival and reproductive success. The lability of
skin pigmentation, in particular, in relation to diverse environmental
conditions has contributed to the ability of hominins to disperse and
adapt to local circumstances over the long temporal span of human
evolution. This facility was critical during most of prehistory when, in

This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction
in any medium, provided the original work is properly cited and is not used for commercial purposes.
© 2021 The Authors. Pigment Cell & Melanoma Research published by John Wiley & Sons Ltd.

Pigment Cell Melanoma Res. 2021;34:707–729.  wileyonlinelibrary.com/journal/pcmr | 707

708 | JABLONSKI
the absence of body coverings or built shelters, naked skin was the
primary interface between the hominin body and the environment.
This review is focused primarily on the evolution of skin pig-
mentation during the history of anatomically modern people, Homo
sapiens. Inferences about the characteristics of the skin and skin pig-
mentation in pre-­sapiens Homo species are provided only in instances
where interbreeding of these species with H. sapiens appears to have
clearly influenced skin pigmentation in descendant populations of
modern people. I have taken an historical and geographic approach
here so that readers will achieve a comprehensive understanding
of how skin pigmentation evolved in groups of people dispersing at
different times into different environments, with different comple-
ments of genes, culturally determined behaviors, and material cul-
tures. Skin pigmentation is one of the most thoroughly researched of
complex phenotypic traits in humans, but this expanded knowledge
base has allowed us to see just how much more needs to be done,
especially in understanding the factors affecting the expression of
skin pigmentation genes. The importance of skin pigmentation as a
human phenotypic trait stems from its biological role in prehistory
and its diverse social meanings in history; this review will focus on
the former, not the latter.
Studying the evolution of human skin pigmentation is challenging
because the trait has been influenced by a changing array of multi-
ple variables over time. Identifying the genes and population genetic
processes influencing human skin pigmentation is important, but
not sufficient, for achieving a comprehensive understanding of the
evolution of the trait. This is because human skin pigmentation is
a highly contingent trait, which has changed under different com-
binations of genetic and cultural conditions over time and through
space. It has been influenced by natural selection, population ad-
mixture, and population bottlenecks as hominins have dispersed into
different biomes at various points in the past, but it has also been
influenced by diverse cultural practices in different places and at
different times in the past. In the last 20,000 years (kya), especially,
skin pigmentation evolved under different combinations of genetic
and cultural factors, as humans have dispersed relatively rapidly into
diverse—­including extreme—­environments. Changes in skin pigmen-
tation probably occurred rapidly in some cases as relatively small
populations with restricted gene pools moved into extreme high lat-
itude or island environments where selective pressures were strong.
The skin pigmentation of some groups was also probably influenced
by repeated bottlenecks and movements over many generations into
diverse environments with different selective pressures, especially
with respect to the nature and intensity of ultraviolet radiation (UVR).
And, unlike other mammals, modern humans at specific places and
times possessed unique patterns of diet, food procurement prac-
tices, body coverings, and use of the built environment that would
have affected skin pigmentation phenotypes differentially to cre-
ate unique and frequently highly localized biocultural adaptations.
While making for considerable complexity, these phenomena render
the study of the evolution of human skin pigmentation fascinating.
Because of these considerations, this review differs from excellent
recent reviews of this subject because it builds on information on
the nature and expression of skin pigmentation genes, the role of
genetic epistasis, population genetics processes, and mechanisms of
melanin production (Crawford et al., 2017; Feng et al., 2021; Hanel
& Carlberg, 2020a; Lona-­Durazo et al., 2019; Norton, 2019; Pavan &
Sturm, 2019; Quillen et al., 2019; Rocha, 2020) and attempts to show
how human skin pigmentation evolved in a complex and continually
contingent manner in relation to changing complements of genes
and changing backgrounds of geography and culture.
2 | N AT U R A L S E LEC TI O N A N D TH E S K I N
O F E A R LY H O M O SA PI EN S
The genus Homo originated about 2.8 million years ago (mya) in
Africa from a representative of the genus Australopithecus (Villmoare
et al., 2015). The emerging consensus view is that the evolution of
mostly naked skin in the human lineage probably occurred quite
early in the history of the genus Homo in order to facilitate the evap-
orative cooling of eccrine sweat during extended periods of physi-
cal exertion in hot environments (Jablonski, 2004; Lieberman, 2011;
Zihlman & Cohn, 1988). The onset and duration of this process are
not known with certainty, but evidence provided by comparative
study of patterns of neutral variation in the MC!R locus argues for a
selective sweep having occurred approximately 1.2 mya ago that fa-
vored MC1R variants producing eumelanin-­rich protective pigmen-
tation on effectively naked skin (Rogers et al., 2004). Polymorphisms
in other genes favoring enhanced eumelanin production may have
been selected for early in the history of the genus Homo (Feng
et al., 2021), but the time of origin of these variants cannot be
fixed with certainty. The evolution of functionally naked skin was
also associated with changes in epidermal barrier functions and the
genes of the epidermal differentiation complex (Elias, 2005; Elias &
Friend, 1975; Goodwin & de Guzman Strong, 2017).
The evolution of dark pigmentation early in the evolution of
the genus Homo has been studied primarily from the perspective
of identifying the nature and action of putative selective factors
that accounted for the phenotype. The association of strong sun-
light with dark skin was appreciated by scholars of Greek antiquity
(Hippocrates, 1849) and examined as a likely cause-­and-­effect re-
lationship in the seventeenth century by Robert Boyle, Benjamin
Franklin, and John Hunter (Klaus, 1973). Recognition of the role
played by UVR in this relationship owes to key insights made by
Walter (1958), who was the first to demonstrate a high correlation
between skin pigmentation and latitude as a proxy for UVR. This
was followed up by studies that established that UVR had a higher
correlation to skin pigmentation than environmental temperature,
humidity, or other physical environmental parameters (Chaplin &
Jablonski, 2002; Roberts & Kahlon, 1976). Critical consideration of
selective factors responsible for the UVR–­skin pigmentation rela-
tionship began with Blum's rejection of skin cancer as the likely pri-
mary driver of increased melanin content of the skin because of its
limited effect on mortality (Blum, 1961). The most important result
of Blum's contribution was his focus on the importance of survival,
JABLONSKI
reproductive success, and mechanisms of natural selection in de-
fining adaptive traits, as opposed to identification of factors with
negligible or unknowable selective value. Most of the hypotheses
advanced for the evolution of skin pigmentation in the last 70 years
have failed in this regard, for reasons fully described and reviewed
elsewhere (Jablonski, 2004; Jablonski & Chaplin, 2000).
Two sets of hypotheses for the evolution of skin pigmentation
that are not focused on the primacy of UVR intensity warrant men-
tion here because they have stressed the importance of eumelanin
in enhancing specific barrier functions of the skin. One set focused
on the antimicrobial action of eumelanin in the epidermis and its
heightened importance in combating parasitic and waterborne
disease agents attacking the skin as being the likely agent for se-
lection of darkly pigmented skin in the tropics (Mackintosh, 2001;
Wassermann, 1965). Another set of hypotheses, championed by Elias
and colleagues, has stressed the importance of increased melaniza-
tion of the skin in enhancing the competence of the epidermal
permeability barrier in desiccating terrestrial environments (Elias,
Menon, Wetzel, & Williams, 2009, 2010; Elias & Williams, 2013).
The explanatory power of these hypotheses to account for known
patterns of human skin pigmentation and, in particular, the distribu-
tion of eumelanin-­rich skin has been questioned on several grounds,
including the fact that the skin of the lips and the volar surfaces of
the hands and feet exhibits a near absence of eumelanin pigmenta-
tion in all people despite being subjected to some of the most rigor-
ous physical and antimicrobial challenges of any skin on the body's
surface (Jablonski & Chaplin, 2013). The weight of current evidence
thus supports the theory that UVR is the primary selective agent
which has influenced the evolution of human skin pigmentation.
The distribution and intensity of the different wavelengths of
UVR vary over terrestrial surfaces according to time of day, sea-
son, geographical latitude, altitude, cloud cover, and other factors
(Diffey, 1991). Individual exposure to UVR depends on the intensity
of the ambient UVR in a given location, the fraction of ambient ex-
posure received at a given anatomical site, and the timing and nature
of outdoor activity (Diffey, 1999). In assessing the effects of UVR on
the skin and weighing the relative importance of these effects on the
evolution of skin pigmentation, it is important to appreciate that hu-
mans in prehistory exhibited different lifestyles and patterns of UVR
exposure than modern people. While this may seem self-­evident, it
is rarely taken into account.
The relative importance of different selective factors in the evo-
lution of human skin pigmentation depends on their potential effects
on reproductive success. A potential selective agent like UVR may
damage connective tissue and DNA, for instance, but if the effects
of the damage do not adversely affect the individual's reproductive
success, or if the number of individuals adversely affected is small,
then its evolutionary impact is diminished. In this connection, the
patterns of life history, longevity, and demographics observed in
modern people cannot be assumed for early members of the genus
Homo or even prehistoric H. sapiens. A recent proposal advancing
the importance of skin cancer as a selective agent in the evolu-
tion of human skin pigmentation that was based on an assumption
| 709
of widespread longevity and the importance of grandparental
care of offspring needs thus needs to be questioned (Osborne &
Hames, 2014). It is also important to choose suitable modern human
models or model systems when framing theoretical discussions of
the evolution of skin pigmentation because choices of inappropriate
model systems result can result in misleading conclusions. An import-
ant case in point is another hypothesis proposing protection against
skin cancer as the primary selective agent favoring the evolution of
permanent dark skin pigmentation. In this study, high rates of fatal
skin cancers in depigmented people with albinism living in high UVR
environments were presented as evidence that the most important
function of eumelanin was protection against UVR-­induced damage
to DNA and its connection to diminished reproductive success and
premature death (Greaves, 2014). This hypothesis was based on the
assumption that ancestral hominins had lightly pigmented or depig-
mented skin and that modern people mostly or entirely lacking eu-
melanin in their integument might a suitable model for the ancestral
human state. The notion that very lightly pigmented people or indi-
viduals with albinism could serve as models for understanding the
selective forces operating on the skin of early hominins is wrong. All
living catarrhine primates, including the closest living ape relatives
of humans, have intact pigmentary systems and can develop faculta-
tive pigmentation through tanning of non-­hairy exposed skin or have
permanently melanized exposed skin (Jablonski & Chaplin, 2014b).
In the human lineage, lightly pigmented skin and depigmented skin
due to albinism are highly derived, not ancestral, conditions. The
manifold protective effects of eumelanin in the skin, especially with
respect to mitigation of UVR damage and prevention of carcino-
genesis, have been thoroughly researched and reviewed elsewhere
(Abdel-­Malek, 2009; Hennessy, Oh, Diffey, et al., 2005; Swope &
Abdel-­Malek, 2018; Tadokoro et al., 2003; Young et al., 2017). More
germane in the context of this review is discussion of the likely selec-
tive value of different degrees of eumelanin pigmentation on health
and potential reproductive success. In this context, we cannot as-
sume that the appearance, genetic composition, sun exposure hab-
its, or UVR skin reactions of humans today are the same as those of
our ancestors in prehistory. Choice of appropriate models for ances-
tral or ancient hominin appearance, physiology, or behavior must be
done with great care and will always involve uncertainty because of
the many kinds of variables involved. Consideration of the evolution-
ary significance of eumelanin-­rich skin pigmentation in the human
lineage must be informed by this principle.
2.1 | Folate-­dependent processes and their role
in the evolution of eumelalin-­rich pigmentation
Research conducted first in the 1970s suggested that eumelanin in
the skin may be protective against degradation of folate (Branda &
Eaton, 1978). Appreciation of the centrality of folate in regulating
DNA synthesis and repair grew rapidly in subsequent years, espe-
cially after the connection between folate deficiencies and the then
common class of birth defects, neural tube defects, became widely
710 | JABLONSKI
recognized on the basis of epidemiological and experimental studies
(Bower & Stanley, 1989; Elwood, 1983; Fleming & Copp, 1998; MRC
Vitamin Study Research Group, 1991). Folate, primarily in the form
of 5-­methyltetrahydrofolate (5-­MTHF), is used at the cellular level
for DNA production, the cysteine cycle, and regulation of homocyst-
eine. Knowledge of the specific actions, enzymatic and genetic regu-
lation, and environmental sensitivity of folate grew quickly in light of
increasing appreciation of the vitamin's extensive clinical significance
(Branda & Blickensderfer, 1993; Giovannucci et al., 1995; Lucock &
Daskalakis, 2000; Lucock et al., 2001; Mastropaolo & Wilson, 1993).
Appreciation of the importance of folate to DNA production and
cell proliferation during embryogenesis and spermatogenesis and
its likely sensitivity to UVR lead to initial formulations of a hypoth-
esis about the protective effect of eumelanin pigmentation with
respect to successful pregnancy outcomes (Jablonski, 1992, 1999).
This work was further elaborated when it became possible to exam-
ine patterns of human skin pigmentation (as provided in reports of
standardized skin reflectance measurements taken from indigenous
peoples) relative to levels of remotely sensed UVR as measured by
the NASA Total Ozone Mapping Spectrometer 7 (TOMS-­7) satellite
(McPeters et al., 1996). The TOMS-­7 and subsequent NASA satellites
revolutionized the study of the effects of UVR on biological systems
because they provided standardized, global, spatially continuous
and gridded data that made it possible to assess the potential for
biological damage due to solar irradiation, given the column ozone
amount and cloud conditions on any day, for most places on earth
(Langston et al., 2017). The demonstration that human skin pigmen-
tation was more highly correlated to UVB than to latitude (Jablonski
& Chaplin, 2000) or environmental parameters such as total solar ir-
radiance, temperature, humidity, or rainfall. Chaplin (2004) indicated
that UVR was the most important environmental determinant of skin
pigmentation.
The nature of the specific protective effect of eumelanin in
the epidermis and its effects of evolutionary fitness depends fun-
damentally on where the pigment is localized in the skin. Different
wavelengths of UVR penetrate the skin to different depths of the
epidermis and dermis, depending on the skin site, the amount and
location of eumelanin, and the degree of keratinization of the skin
(Meinhardt et al., 2008). Eumelanin is concentrated within keratino-
cytes in the stratum basale of the epidermis and in this position can
afford considerable protection against damage by UVR, especially
UVB (Fajuyigbe et al., 2018; Fajuyigbe & Young, 2016). The stratum
corneum itself affords some protection against UVB, but the degree
of protection afforded depends on the thickness of individual layers,
the number of layers, the degree of hydration of epidermis (Bruls
et al., 1984). The protective effect of stratum corneum itself is more
significant with respect to UVB shielding in skin with less eumela-
nin content, but the magnitude of this effect is variable and, overall,
minor compared to that of eumelanin itself (Hennessy et al., 2005;
Kaidbey et al., 1979; Olivarius et al., 1997).
Most of the attention paid to the UVB shielding effects of eu-
melanin has been concerned with the sparing of damage to DNA
and mitigation of carcinogenesis because of the pigment's significant
UVR absorptive abilities, which slow the formation of highly muta-
genic cyclobutane pyrimidine dimers (CPD) (Fajuyigbe et al., 2018).
The localization of most eumelanin in the stratum basale also serves
to protect the capillary bed of the dermis from UVR, especially UVB.
Penetration of UVA into the stratum basale is considerably greater
and potentially more harmful in lightly pigmented skin (Meinhardt
et al., 2008). Attention in this connection has been paid primarily to
the cosmetic effects of UVA on the breakdown of connective tissues
in the dermis (Battie et al., 2014), but the demonstrated effects of
UVA on vasodilation of arteries in the capillary bed of the dermis (Liu
et al., 2014) suggest that control of blood pressure may be of more
fundamental importance.
Evidence for the hypothesis that permanent dark pigmentation
in hominin skin was an adaptation to protect against UVR-­induced
degradation of folate in the skin was originally based on direct
effects on fertility such as potential embryo loss or faulty sper-
matogenesis (Jablonski & Chaplin, 2000). These effects are hard
to demonstrate in living human subjects or through retrospective
epidemiological studies. Folate levels and folate metabolism are
affected by primary folate deficiency, non-­folate B-­vitamin nutri-
ent deficiencies, and genetic variations that influence cellular folate
accumulation and utilization (Perry et al., 2004; Stover, 2009), and
these factors were generally not considered in 20th-­century sur-
veys of folate levels within and between populations. The clearest
epidemiological demonstration of the possible folate-­
sparing ef-
fect of dark pigmentation against UVR challenge came from a study
demonstrating the lower prevalence of NTDs in darkly pigmented
as opposed to lightly pigmented South Africans, despite the greater
affluence and presumed better diet of the latter group (Buccimazza
et al., 1994). Experimental studies of the effects of UVA and UVB on
serum and red cell folate conducted in vitro and in vivo have yielded
mixed results and have been difficult to interpret because of incon-
sistencies in experimental conditions and folate assay standardiza-
tion (Borradale et al., 2014; Borradale & Kimlin, 2012; Fukuwatari
et al., 2009; Gambichler et al., 2001; Juzeniene et al., 2009; Wolf
& Kenney, 2019), but most studies have found depletion of folate
to varying degrees, depending on the wavelength and duration of
UVR exposure, the pre-­exposure folate level, and the species of fo-
late being assayed. Experimental demonstration of a decline in epi-
dermal 5-­MTHF following UVA exposure in individuals with lightly
pigmented but not darkly pigmented skin (Hasoun et al., 2015) is
potentially more significant because it indicates the importance of
UVR-­induced folate depletion in the skin after a short UVR exposure
and possible systemic depletion following prolonged exposure.
Appreciation of the central importance of folate on human health
through its effects of DNA synthesis and methylation (Stover, 2009)
has been augmented in the last 20 years by research demonstrat-
ing direct effects of folate on vascular function. These effects are
pertinent to the evolution of skin pigmentation because of the role
played by folate (as 5-­MTHF and as its further derivative, tetrahydro-
biopterin, or BH4) in improving nitric oxide (NO)-­mediated endothelial
function and affecting vasodilation, blood pressure, and thermoregu-
lation (Alexander et al., 2013; Ng et al., 2009; Stanhewicz et al., ,2012,
JABLONSKI
2015). UVA triggers cutaneous vasodilation, probably by releasing
NO from preexisting stores of nitrite in the dermis (Liu et al., 2014).
The discovery that lower NO levels contributed to attenuated vaso-
dilation in response to local heating of the skin of older individuals
and that these responses could be restored by folate administra-
tion (Kenney, 2017) led to studies aimed at discovering the likely
molecular mechanisms whereby folate improved NO bioavailability
and protected endothelial function (Stanhewicz & Kenney, 2017).
Recent findings indicating that acute UVB exposure attenuated NO-­
mediated vasodilation of the cutaneous microvasculature via degra-
dation of 5-­MTHF led to the pursuit of studies examining the effects
of skin pigmentation, age, and folate status on vasodilation and ther-
moregulation (Wolf & Kenney, 2019). Available evidence indicates
that folate degradation in response to UVR exposure is likely dose-­
dependent and requires relatively large doses, repeated prolonged
exposures, or both to observe reductions in serum or red blood cell
folate (Wolf & Kenney, 2019). These environmental conditions would
have been met in the early history of the genus Homo when hominins
were active in the sun or at least exposed to strong, equatorial sun-
light for most daylight hours without the benefit of body coverings.
The sum of the effects of UVR-­induced folate loss on hominins
cannot be accurately estimated, but there is little doubt that low-
ered fertility due to embryo loss and impaired spermatogenesis
combined with impairments of vasodilation and temperature regu-
lation would have represented direct and severe selective pressures
favoring conservation of folate through increased eumelanin in the
functionally naked skin of early Homo. This does not mean that early
Homo had “dark skin,” but that selection favoring enhanced eumel-
anin pigmentation in the skin was evolving early in the evolution
of the genus, along with polymorphisms in genes affecting folate
metabolism. Because folate metabolism is regulated by several en-
zymes, including methylenetetrahydrofolate reductase (MTHFR),
methionine synthase (MTR), and methionine synthase reductase
(MTRR), and because the activity of these enzymes varies according
to different functional polymorphisms, these polymorphisms appear
to have evolved in response to seasonal availability of folate and lev-
els of UVR (Jones et al., 2018; Lucock et al., ,2010, 2017). It is thus
probable that multiple mechanisms, including enhanced eumelanin
pigmentation and conservation of folate through modification of en-
zymatically controlled metabolism, evolved in the face of seasonally
dependent variations in folate availability and UVR levels.
2.2 | Vitamin D and skin pigmentation
The importance of vitamin D in connection with the evolu-
tion of human skin pigmentation was first introduced by Murray
(Murray, 1934) and later elaborated by Loomis (Loomis, 1967). These
were based, at that time, on the recognition of the physiological im-
portance of vitamin D in the growth and maintenance of the skeleton
and understanding that production of vitamin D could be catalyzed
only by specific wavelengths of UVR, from about 290–­320 nm, in the
UVB range. Loomis originally opined that “white skins” had evolved
| 711
to maximize cutaneous photoconversion of 7-­dehydrocholesterol (7-­
DHC) into vitamin D under low UVB conditions at high latitudes and
that “black skins” had evolved to protect against vitamin D toxicity
due to potential overproduction of vitamin D at low latitudes under
high UVB conditions (Loomis, 1967). Research into the mechanisms
of cutaneous vitamin D synthesis by Holick and colleagues provided
evidence that the process was tightly photochemically controlled
and that the eumelanin content of the skin determined the rate of
conversion of 7-­DHC to previtamin D3 (pre-­D). (Clemens et al., 1982;
Holick et al., 1980, 1981). This body of research demonstrated that
increased eumelanin content of the skin could not be implicated in
preventing vitamin D toxicity, but that depigmented skin facilitated
cutaneous vitamin D production because of the fact that eumelanin
competes with 7-­DHC for UVB photons (Holick et al., 1981). This
research laid the groundwork for subsequent studies establishing
the nature and genetic basis of the selective forces acting on the
evolution of skin pigmentation in populations dispersing into and
inhabiting regions of lower and more highly seasonal UVB (Beleza,
Santos, et al., 2013; Gozdzik et al., 2008; Hanel & Carlberg, 2020a;
Izagirre et al., 2006; Jablonski & Chaplin, 2000, 2010; Makova &
Norton, 2005).
The body of research pertaining to the importance of vitamin D
in bone metabolism and many cellular and immunological processes
is now immense and is reviewed elsewhere (Lips, 2006, 2007; Lips
et al., 2014; Sassi et al., 2018). Issues raised about the strength of
natural selection acting to reduce eumelanin pigmentation in order
to enhance cutaneous vitamin D synthesis (Elias et al., 2009; Elias &
Williams, 2013, 2016; Robins, 2009) have been countered by con-
siderable evidence. The claim that loss of eumelanin and enhance-
ment of cutaneous vitamin D production did not significantly improve
the reproductive success of hominins dispersing to high latitudes
(Robins, 2009) was called into question by challenging incorrect as-
sertions, including the claim that, even at the highest latitudes, stored
vitamin D alone is sufficient to meet physiological needs during
months when the UVB in sunlight is insufficient to catalyze cutaneous
vitamin D production (Chaplin & Jablonski, 2009, 2013). Other claims
centering around reduction in epidermal eumelanin having been se-
lected for altered epidermal barrier functions have also been found
deficient. These have focused on the significance of reduced levels of
the protein filaggrin in the stratum corneum, leading to enhanced cu-
taneous synthesis of pre-­D and also on the argument that reduction
in eumelanin production was favored by natural selection because of
the importance of conserving energy (Elias & Williams, 2013). These
explanations failed to demonstrate any significant evolutionary ad-
vantage for reduction of filaggrin (Jablonski & Chaplin, 2013), and
further study of variation in loss of function genes affecting filaggrin
in the epidermis failed to demonstrate a relationship between genetic
variants and latitude (as a surrogate for UVB and cutaneous vitamin D
synthesis potential) (Eaaswarkhanth et al., 2016).
A key consideration in understanding the evolution of skin pig-
mentation in people living under high and less seasonally variable
UVR conditions—­generally within the tropics—­is the degree to which
eumelanin pigmentation actually slows the cutaneous biosynthesis
712 | JABLONSKI
of vitamin D. Recent experimental work done by Young and col-
leagues on human subjects has demonstrated that darkly pigmented
individuals (Fitzpatrick type VI according to study protocols) exhib-
ited a melanin vitamin D inhibition factor of 1.3–­1.4 relative to lightly
pigmented individuals (Fitzpatrick type II) (Young et al., 2020). They
reasoned that this significant, but relatively modest, inhibitory ef-
fect may have been due to the fact that photoconversion of 7-­DHC
into pre-­D occurs mostly in the stratum granulosum and stratum spi-
nosum of the epidermis, above the heavily melanized layers of the
basal epidermis (Young et al., 2020).
Vitamin D serves many critical functions in the body, and de-
ficiency in the vitamin increases susceptibility to a range of devel-
opmental, chronic, and infectious diseases via its effects on the
epigenome and on the expression of many genes on nearly all organs
and tissue types in the body (Bora & Cantorna, 2017; Bustamante
et al., 2020; Caccamo et al., 2018; Carlberg, 2019; Sassi et al., 2018).
The recognition that vitamin D improves endothelial function by
signaling for the transcription of endothelial nitric oxide synthase
(eNOS) and thereby acting to preserve production of NO and
healthy peripheral vascular function augments our understanding of
the vitamin's consummate importance in human physiology and has
potentially great consequences for our understanding of the evolu-
tion of skin pigmentation (Wolf et al., 2020; Wolf & Kenney, 2019).
Vitamin D is produced in the upper epidermis through UVB-­induced
catalysis of 7-­DHC into pre-­D, and it is required in the dermis to
preserve NO production required for healthy vasodilation. This im-
portant function of vitamin D probably accounts for the intriguing
finding of reduced all-­cause mortality in a large cohort of lightly pig-
mented Scandinavian women reported by Lindqvist and colleagues
(Lindqvist et al., 2020). The role of vitamin D’s role in eNOS signaling,
control of vasodilation, and regulation of blood pressure warrants
further investigation in an evolutionary context with respect to the
evolution of skin pigmentation under low UVR conditions (Wolf
et al., 2020). It is also of potentially great relevance in clinical con-
texts with respect to the importance of low and carefully controlled
doses of natural UVR sufficient to produce vitamin D and maintain
healthy vasodilation and blood pressure (Alfredsson et al., 2020;
Wolf et al., 2020). This consideration is of particular concern in ha-
bitually indoor-­dwelling people, older people with attenuated cuta-
neous vitamin D production abilities, and darkly pigmented people
living in low UVR environments.
The manifest importance of vitamin D in human physiology im-
plies that complex, genetically based mechanisms for establishing
and maintaining vitamin D sufficiency have evolved in the course
of human evolution. Modification of the production and packaging
of eumelanin in the skin affects the penetration of UVB into the
epidermis and the physical potential for photoconversion of 7-­DHC
into pre-­D, but levels of vitamin D can and are affected by many
other processes and pathways. Advances in genomics and archeog-
enomics have made possible a significant shift in our understanding
of these processes in recent years, to the extent that there is now
greater appreciation of how the evolution of genetic variants associ-
ated with vitamin D metabolism and signaling has evolved in concert
with skin pigmentation. As modern populations have dispersed and
undergone bottlenecks and admixture, variant genes encoding for
proteins responsible for photoconversion of 7-­DHC into pre-­D (Kuan
et al., 2013) and governing transport, metabolism, and signaling of
vitamin D, including DHCR7, GC, CYP2R1, and CYP24A1 (Hanel &
Carlberg, 2020a, 2020b), have undergone changes in frequency
appeared and become common. Genetic changes affecting vitamin
D metabolism rather than skin pigmentation have been particularly
important (Hanel & Carlberg, 2020a, 2020b), especially at extreme
northern high latitudes where levels of UVB are low and highly sea-
sonal, creating conditions for only short and sporadic cutaneous
photosynthesis of vitamin D (Chaplin & Jablonski, 2013; Jablonski
& Chaplin, 2010). Lastly, the nature of the genetic changes occur-
ring was being mediated by numerous lifestyle variables (includ-
ing diet, typical body coverings and kinds of shelter, and patterns
of daily activity) which affected vitamin D status and would have
contributed to individual survival and reproductive success. Thus,
consideration of the contingent nature of biocultural compromises in
different places and at different times is essential to the study of the
evolution of human skin pigmentation and human health (Chaplin
& Jablonski, 2013). Genetically driven changes in skin pigmentation
favoring less integumental eumelanin were only one factor in the
“adaptive equation” contributing to healthy vitamin D status.
2.3 | Likely skin pigmentation of early Homo sapiens
The probable loss of most body hair during the early evolution of the
genus Homo left the body highly vulnerable to the effects of UVR,
as well as to many other potentially harmful physical, chemical,
and biological agents. The evolution of protective eumelanin-­rich
constitutive pigmentation in the tropical-­dwelling, African ances-
tors of all modern people was probably a step-­by-­s tep process in-
volving many genetic loci. Early in the history of skin pigmentation
genomics, considerable attention was focused on the primacy of
a selective sweep affecting the MC1R locus to eliminate variation
and establish permanent dark pigmentation in the human lineage
(Rana et al., 1999; Rogers et al., 2004). Since those early studies,
the field of skin pigmentation genomics has been revolutionized
by the development of high-­throughput sequencing technologies
coupled with the application of genome-­wide association studies
(GWAS) for identifying multiple genes associated with human skin
pigmentation and the execution of genome-­wide scans of natural
selection. These developments have made possible the identifica-
tion of many genes contributing to skin pigmentation, and the de-
gree to which they have been acted upon by natural selection (Feng
et al., 2021). For many loci of interest, application of coalescent
studies on specific derived alleles has made possible estimation of
when was the most recent common ancestor of existing derived
alleles first occurred.
Based on these studies, it is possible to estimate that multiple
derived alleles contributed to the evolution of dark pigmentation in
ancestral H. sapiens. These include two derived alleles of the major
JABLONSKI
facilitator superfamily domain-­
containing protein 12 (MFSD12),
which appeared at or near the time of origin of H. sapiens, about
0.3 mya (Crawford et al., 2017; Feng et al., 2021). Other candidate
loci, including multiple alleles of the DNA damage-­binding protein
1 (DDB1), may also have been present in early H. sapiens because
of their presence at high frequencies in modern African and in
some non-­European populations today (Crawford et al., 2017; Feng
et al., 2021) (Figure 1), thus suggesting that derived alleles were
carried in one of the populations that dispersed along the southern
and southeast Asian coasts and into Melanesia. Detailed examina-
tion of the geographical pattern of variation in one of these alleles,
rs7948623 (T), further suggests that it may have been under positive
selection, resulting in further skin darkening, in regions of the world
including East Africa, parts of South Asia, and Melanesia with ex-
tremely high environmental UVR (Feng et al., 2021). The key point
is that the ancestor of all H. sapiens had darkly pigmented, but not
necessarily maximally darkly pigmented, skin (Chaplin, 2004; Hanel
& Carlberg, 2020a). In Africa today, and among many South Asians
and Melanesians, there is great variation in the darkness of “darkly
pigmented” skin that appears to be due to variations in UVR inten-
sity (Chaplin, 2004; Jablonski & Chaplin, 2014a). Genomic studies
of the last two decades have revealed that the genetic “palette” of
skin pigmentation gene variants is great and that many combinations
of multiple variant genes have contributed to the complex pattern
of skin pigmentation phenotypes and genotypes observed today.
Under more highly seasonal UVR regimes, the evolution of enhanced
tanning abilities and genes contributing to rapid development of fac-
ultative pigmentation has been favored (Quillen et al., 2019). Despite
the manifest importance and relevance of tanning genetics to skin
cancer dynamics and skin esthetics, relatively little genomics-­based
research on the topic has been done, especially in African and in-
digenous American populations. Although advances in evolutionary
skin pigmentation genetics have been enormous, there is an urgent
need to validate candidate skin pigmentation variants using GWAS
and scans of natural selection, using functional experiments in vitro
and in vivo (Feng et al., 2021).
F I G U R E 1 The “hairy timeline of human evolution” illustrating, schematically, the evolution of body hair and skin color in pre-­Homo
sapiens members of the human lineage. Prior to the evolution of early members of the genus Homo, about 2.8 mya, hominin skin, was lightly
pigmented (but tannable) and covered with dark hair. The evolution of mostly hairless bodies beginning about 2 mya inaugurated a selective
sweep eliminating MC1R polymorphism, after which time selection for dark pigmentation variants of DDB1 and MFSD12 persists through the
time of origin of H. sapiens, about 0.3 mya
| 713
3 | S K I N PI G M E NTATI O N E VO LU TI O N I N
R E L ATI O N TO D I S PE R SA L S , G E N E S , A N D
CU LT U R E I N PR E H I S TO RY
The processes affecting the evolution of human skin pigmenta-
tion have changed in relative importance over time. In the last
100,000 years (100 kya), H. sapiens has gone from being an exclu-
sively African species to a global one. The nature and speed of major
dispersals of modern humans varied according to many geographic,
demographic, technological, and cultural variables. With gains in tech-
nological competence and, especially, with the domestication of ani-
mals used for food and transportation, humans became more mobile
and had better abilities to buffer themselves against the exigencies
of the environment because they could control more aspects of per-
sonal nutrition and bodily protection through technology and cultural
practices. Through time, and especially since the beginning of history
sensu stricto about five thousand years ago, culturally based prefer-
ences for skin color (including sexual selection and social selection)
have also influenced regional trends in the skin pigmentation. For all
of these reasons, there has probably never been a time or place in the
history of our species when skin pigmentation has been in equilibrium.
The following review of the history of skin pigmentation in
the major regions of the world is, perforce, speculative because of
the multiplicity of variables being considered. The level of detail
presented here falls far short of that covered in global reviews of
human dispersals that have examined the details of human move-
ments from the perspectives of genomics, genetics, and population
genetics (James et al., 2019; Nielsen et al., 2017; Posth et al., 2016).
Nonetheless, they form a basis for discussion and future research.
3.1 | Africa
The earliest history of H. sapiens occurred in Africa and has been
reconstructed through a combination of sparse fossil evidence and
inferences from archeogenomics (Galway-­Witham & Stringer, 2018;
714 | JABLONSKI
Henn et al., 2018; Hublin et al., 2017; Stringer, 2016; White
et al., 2003). The skeletal traits which define H. sapiens emerged in
Africa over the course of about 100,000 years, from 0.3 to 0.2 mya
(Galway-­Witham & Stringer, 2018). By 0.2 mya, diverse forms of
anatomically modern humans lived in Africa, mostly in dispersed
regional and environmental enclaves, and pursued technologi-
cally and artistically complex but locally distinctive cultures (Scerri
et al., 2018). The genetic history of modern people in Africa thus
reflects deep roots, and complex patterns of isolation and reticula-
tion. Some of the reasons for this can be readily appreciated when
the vastness, ecological diversity, and remarkable latitudinal extent
F I G U R E 2 Generalized timeline of
main events in the evolution of skin
pigmentation in Africa, by region. After
originating around 0.3 mya, H. sapiens
evolved in Africa only until about 80 kya
when some dispersals from northeastern
Africa into the Afro-­Arabian Peninsula
occur. (a) In East Africa, continued
purifying selection on MC1R variants is
accompanied by positive selection on
variants of DDB1 and MFSD12 leads to
darker pigmentation. The introduction
of the classic depigmentation SLC24A5
variant around 9 kya via the Afro-­
Arabian Peninsula sees its rapid dispersal
southward. (b) In West Africa, purifying
selection on MC1R variants continues,
accompanied by positive selection on
variants of DDB1 and MFSD12 producing
darker pigmentation. (c) In South Africa,
among Khoe-­san hunter-­gatherers,
relaxation of selection on MC1R is
accompanied by positive selection on the
SLC24A5 introduced from northeastern
Africa. The dispersal of darkly pigmented
Bantu-­language speaking agriculturalists
from western and central Africa from
5 ka onward and their admixture with
Khoe-­san hunter-­gatherers leads to
the evolution of complexly mixed
pigmentation genotypes and a wide range
of moderately pigmented phenotypes
of Africa are considered. Other reasons are demographic. Until the
advent of agriculture and animal husbandry in Africa about 5 kya,
most populations of hunter-­gatherers were relatively small, scat-
tered, and probably experienced repeated bottlenecks and periods
of expansion due to sometimes dramatic environmental perturba-
tions (Ambrose, 1998; Hsieh et al., 2016; Powell et al., 2009).
In the specific context of human skin pigmentation evolution in
Africa, the diversity of UVR regimes in the African continent, includ-
ing the highly seasonal pattern of UVB in the far south, is one of many
factors that must be appreciated (Coussens et al., 2015; Jablonski &
Chaplin, 2014a). These patterns are reflected in the complex mosaic
JABLONSKI
of genes that contribute to skin pigmentation across Africa today
(Crawford et al., 2017; Feng et al., 2021; Lin et al., 2018; Martin
et al., 2017; Rocha, 2020). The strong and relatively seasonally in-
variant UVR regimes of East Africa, including the Horn of Africa and
the East African coast, have continued to drive selection for increas-
ingly dark skin pigmentation in these regions (Chaplin, 2004; Feng
et al., 2021; Jablonski & Chaplin, 2000, 2014a). This trend has also
been influenced by the wearing of relatively little clothing during
daylight hours and while active, up to and including historical times,
because of extreme environmental heat. A trend toward increas-
ing pigmentation darkening also appears to have occurred in West
Africa, but the combination of genetic variants contributing to this
is less clear (Crawford et al., 2017; McEvoy et al., 2006). The nature
of the genes and genetic epistasis contributing to the enhancement
of dark constitutive pigmentation and tanning abilities in equatorial
African people (and elsewhere) is still poorly understood, but it is
likely that insights will come from functional genomic studies and
those focused on the study of recently admixed populations in order
to determine causal effects (Beleza, Johnson, et al., 2013; Lona-­
Durazo et al., 2019; Rocha, 2020).
The other major phenomena that have influenced the evolu-
tion of skin pigmentation in Africa were recent human dispersals,
but not those associated with European colonialism. The first of
these was the dispersal of people from Eurasia via the Afro-­Arabian
Peninsula into eastern and southern Africa. These people carried
derived variants of SLC24A5 (solute carrier family 24 [sodium/potas-
sium/calcium exchanger], member 5) associated with depigmented
skin (Martin et al., 2017). These variants spread relatively quickly
southward, beginning around 5,000 yr (Crawford et al., 2017), and
were favored under positive selection in the far southern latitudes
of Africa among Khoe-­san hunter-­gatherers and pastoralists, espe-
cially in the last 2,000 yr (Lin et al., 2018; Martin et al., 2017). The
evidence for positive selection for SLC24A5 variants that conferred
some skin lightening in the indigenous Khoe-­san peoples inhabiting
the lower and more seasonal UVR conditions of southern Africa is
significant. It complements evidence that non-­
synonymous mu-
tations in the MC1R locus exist in the Khoe-­san (John et al., 2003)
and that other variant forms of MC1R may have arisen in southern
African under relaxed selection. It also illustrates that a new genetic
variant can achieve high frequency in human populations after being
introduced if it affects a trait that positively impacts fitness. This
result is also important because it puts paid to arguments that the
relatively light skin pigmentation of the Khoe-­san was due to genetic
admixture with Europeans early in colonial history.
The second major dispersal event within Africa that has affected
the distribution of skin pigmentation phenotypes and the continu-
ing evolution of skin pigmentation by population admixture is the
expansion of Bantu-­language speakers from western Africa into
central, eastern, and southern Africa, beginning about 5.6 kya (Li
et al., 2014; Patin et al., 2017). The timing and nature of dispersals
of Bantu-­speaking peoples have been characterized using genome-­
wide microsatellite markers and are now understood to have, in gen-
eral, proceeded from west to east, and then south (Li et al., 2014).
| 715
Linguistic, archeological, and genetic evidence for the movement
of Bantu-­speaking agriculturalists attests that this was one of the
largest and most genetically impactful dispersals in human history
because of its effects on preexisting populations of rainforest-­and
desert-­dwelling hunter-­gatherers in western, central, and southern
Africa (Patin et al., 2017). With respect to skin pigmentation, the in-
gress of very darkly pigmented people into southern Africa in recent
millennia appears to have adversely affected vitamin D status and
health, especially in modern, mostly urban populations in southern-
most Africa (Coussens et al., 2015). A hypothetical skin pigmentation
timeline for the major regions of Africa is presented in Figure 2.
3.2 | Coastal south and southeast Asia,
Melanesia, and Australia
The timing and routes of the dispersal of H. sapiens into Eurasia, and
the population sizes of dispersing populations, have been studied in-
tensively by geneticists and genomicists in the last 20 years and are
now reasonably well understood (Henn et al., ,2012, 2016; Nielsen
et al., 2017; Pagani et al., 2016; Reyes-­Centeno et al., 2014). The
number, timing, and exact routes of these dispersals are still de-
bated, but genetic and paleontological evidence indicates that the
first groups of H. sapiens dispersed out of Africa beginning about
70 kya, following a primarily coastal route from the Afro-­Arabian
Peninsula into south and southeast Asia, thence into Melanesia and,
eventually, into Australia (Groucutt et al., 2015; James et al., 2019;
Malaspinas et al., 2016; Pagani et al., 2016; Posth et al., 2016; Reyes-­
Centeno, 2016; Reyes-­Centeno et al., 2014). Divergence of popula-
tions ancestral to modern Papuans and Aboriginal Australian from
Eurasians is estimated from genomic evidence to have occurred
72–­51 kya, after which time admixture of ancient Denisovans with
an ancestral Papuan-­Australian population occurred (Malaspinas
et al., 2016).
Populations dispersing and living along the coasts of southern
and southeastern Asia in the Late Pleistocene experienced strong
and seasonally relatively invariant UVR levels, favoring maintenance
of dark pigmentation, probably involving selection on ancestral gene
variants MFSD12 and DDB1 (Feng et al., 2021) and variant forms of
SLC24A5 and OCA2 (Jinam et al., 2017; Stokowski et al., 2007). The
contribution of Denisovan genes to these populations is recognized
(Gittelman et al., 2016; Jinam et al., 2017) but the genetic contri-
bution of Denisovans to skin pigmentation is not yet clearly estab-
lished. In considering the evolution of skin pigmentation in these
populations, the effects of population size, diet, and habitual raiment
must be considered, even if the strength of these influences cannot
be quantified easily. The populations dispersing along these coasts
were hunter-­gatherers and probably of small population size. Some
of these, notably several so-­called “Negrito” populations, manifest
genetic evidence of severe population bottlenecks as well as recent
admixture (Jinam et al., 2017). Based on ethnographic accounts of
living Andamanese, these people ate traditional diets of fish, wild
boar, shellfish, turtle, and turtle eggs, along with foraged tubers and
716 | JABLONSKI
fruits (Headland, 1989; Sahani, 2010). They also wore few, if any,
body coverings. This lifestyle conduced to a high level of vitamin
D sufficiency, thereby rendering unlikely the development of any
level of vitamin D insufficiency as long as a traditional diet was being
consumed. This may have released a selective constraint on further
pigment darkening.
Knowledge of the skin pigmentation phenotypes and pigmen-
tation genes of Melanesians and Australian Aboriginals is limited,
but the available data indicate that skin pigmentation is very dark,
with Bougainvilleans exhibiting the darkest levels of pigmentation
measured by reflectometry among people living today (Jablonski
& Chaplin, 2000; Norton et al., 2006). Similar, extremely dark
pigmentation reportedly existed among Aboriginal Tasmanians
(Diamond, 2005). Melanesian populations notably lack evidence
for purifying selection at the MC1R locus (Norton et al., 2015) and,
thus, their extremely dark pigmentation must be accounted for by
enhanced eumelanin production and tanning abilities controlled by
other genes, possibly MFSD12 and DDB1, and others. Under these
conditions, dark skin pigmentation evolved to what appears to be
a threshold level, past which no further darkening appears to be
possible (Chaplin, 2004). It is noteworthy that the people who ex-
hibit some of the darkest skin pigmentation observed are not only
exposed to extremely high environmental UVR, but, traditionally,
wear few clothes and eat diets rich in fish. These are also mostly
coastal and island populations that have also been relatively isolated
throughout most of history because of lack of accessibility over
land. Thus, they were not subject to high levels of admixture from
mainland groups. These populations are exemplars of one of the
extremes of skin pigmentation evolution, occurring as the result of
people being isolated in geographic dead-­ends, while simultaneously
experiencing little admixture and positive selection. In these cases,
diets replete in vitamin D may have worked to further promote the
evolution of increasingly dark, protective eumelanin pigmentation
up to a threshold level. This hypothesis invites further study of genes
affecting skin pigmentation, vitamin D metabolism, and the eumel-
anin threshold effect. A hypothetical skin pigmentation timeline for
coastal South and Southeast Asia, Melanesia, and Australia is pre-
sented in Figure 3.
3.3 | Hinterland Eurasia and the Americas
The evolution of skin pigmentation in Eurasia, especially in Europe,
has been the focus of considerable attention (Norton, 2019; Quillen
et al., 2019), especially with the advent of archeogenomics and
the functional genomic interpretation of skin pigmentation genes
in ancient Eurasian populations. New and comprehensive reviews
of Eurasian skin pigmentation genomics in relation to historical
genomics should be consulted for details, especially with regard
to the movements of peoples and genes in historic times and their
likely effects on skin pigmentation (Allentoft et al., 2015; Hanel &
Carlberg, 2020a; Ju & Mathieson, 2020; Røyrvik et al., 2018). Here,
I reiterate salient points of those reviews and other works and sup-
plement them sparingly with some additional insights.
Inferences about the likely evolutionary factors affecting human
skin pigmentation among people living at high latitudes in Eurasia
F I G U R E 3 Generalized timeline of
main events in the evolution of skin
pigmentation in Coastal South and
Southeast Asia, Melanesia, and Australia.
(a) Beginning around 70 kya, small
populations of coastal dispersers make
their way along the coasts of southern
and southeastern Asia. Introgression from
Denisovans occurs about 43 kya, possibly
involving introduction of variants favoring
darker pigmentation. (b) In Melanesia
and Australia, following the divergence
from mainland coastal groups, continued
positive selection on variants of DDB1,
MFSD12, and other loci promotes in
the absence of purifying selection on
MC1R produces dark and very dark skin
pigmentation phenotypes
JABLONSKI
have been mooted in the literature for nearly a century (Loomis, 1967;
Murray, 1934). Emphasis on loss of pigmentation in order to promote
cutaneous vitamin D photosynthesis at high latitudes has been em-
phasized, but so too has the importance of consumption of vitamin
D-­rich foods in regions where cutaneous biosynthesis is insufficient
to fulfill year-­round needs for the vitamin (Chaplin & Jablonski, 2013;
Jablonski & Chaplin, 2000, 2010). Elucidation of the genetic basis for
depigmentation began with the landmark study in which a zebrafish
model was used to demonstrate the action of the SLC24A5 variant
common to northwestern European people (Lamason et al., 2005).
The absence of the same variant in East Asian peoples living at sim-
ilar latitudes (Lamason et al., 2005; Norton et al., 2007) inaugurated
an intensive search for the genes responsible for depigmentation in
East Asians. Subsequent historical genomic studies of modern and
ancient populations have shown that depigmentation in Europeans,
especially among far northern-­dwelling peoples, occurred in a step-­
wise fashion, beginning with a shared variant of the Kit ligand (KITLG)
gene in the common ancestor of western European and east Asian
people (Hanel & Carlberg, 2020a; Lao et al., 2007; Sulem et al., 2007).
Extreme depigmentation in northwestern Europeans involved mul-
tiple skin pigmentation genes and appears to have been further pro-
moted by the introduction of agriculture (Brace et al., 2019). At the
highest European latitudes, including Scotland, genetic variants con-
tributing to mostly depigmented or lightly pigmented skin comprise
one component of the biocultural compromise necessary for survival
and reproductive success under low and markedly seasonal UVB at
extreme high latitudes (Chaplin & Jablonski, 2013). Modifications
of genes affecting the production and metabolism of vitamin D
constitute another part of this compromise, in some cases offset-
ting extreme depigmentation in high-­latitude Eurasian populations
(Hanel & Carlberg, 2020a). The last component of the high-­latitude
biocultural compromise is a vitamin D-­rich diet, focused on oily fish
and, depending on the location, also including marine mammals and
wild or domesticated reindeer (Chaplin & Jablonski, 2013; de Barros
Damgaard et al., 2018; Ross et al., 2006). The fragility of the bio-
cultural compromise at high latitudes in Eurasia is illustrated by the
high prevalence of chronic lifestyle diseases, including cardiovascu-
lar disease and metabolic syndrome, in populations that mostly or
completely abandon vitamin D-­rich diets (Chaplin & Jablonski, 2013;
Ross et al., 2006).
The evolutionary trend toward depigmented skin in high-­latitude
Eurasian populations involved numerous genes affecting skin pig-
mentation and vitamin D metabolism and did not result in uniform
adaptive strategies across the great expanse of northern Eurasia fa-
voring extreme depigmentation (Hanel & Carlberg, 2020a, 2020b).
Rather, what we observe is that skin pigmentation has evolved as
part of a larger genetic and cultural package favoring enhanced
availability and economical utilization of dietary and cutaneously
synthesized vitamin D. Because habitation of far-­northern latitudes
in Eurasia was predicated on the development of material culture,
including sewn clothing, which afforded protection against extreme
cold and wind, the surface area of skin available for cutaneous vita-
min D production was reduced, thereby intensifying the selective
| 717
pressure for depigmentation. A further consideration is that in-
creased thickening of the stratum corneum in response to repeated
outdoor conditions and UVR (Oh et al., 2004) would also have
served to attenuate any possible cutaneous vitamin D production
under these conditions (Young et al., 2020).
In far northwestern Europe, the extreme physical isolation of
people in northernmost Britain at the end of the Pleistocene and
early Holocene favored maximal skin depigmentation and a near ab-
sence of eumelanin in the skin. This was made possible by positive
selection for the classic depigmentation variant of SLC24A5, and by
relaxation of selection pressure on the MC1R locus, resulting in high
levels of polymorphism at the locus (Harding et al., 2000; Latreille
et al., 2009; Rees, 2000). Under these circumstances, cutaneous
production of vitamin D could be maximized during the few months
when photocatalytic UVB wavelengths were present in the sunlight
(Chaplin & Jablonski, 2013; Jablonski & Chaplin, 2000, 2010). During
the millennia in prehistoric and early historic times when people did
not travel widely or migrate to other climes, this highly depigmented
skin phenotype worked well. Because their UVR regimes were mark-
edly different from those of people in the tropics, they experienced
only short periods of UVB during the height of the summer and sun-
burns would have been rare (Chaplin & Jablonski, 2013; Jablonski
& Chaplin, 2010). Outdoor lifestyles meant that people adapted
to gradually changing durations and intensities of UVR—­
mostly
UVA—­exposure throughout the year, to which their skin adapted
primarily by thickening of the stratum corneum; the fact that they
could develop only negligible protection from melanin production
did not matter because the challenge from UVB was not severe
or prolonged (Bech-­Thomsen & Wulf, 1995; Hennessy, Oh, Rees,
et al., 2005; Sheehan et al., 1998). Under these conditions, mostly
depigmented skin was not a liability because it did not detract from
health and well-­being and did not affect reproductive success. This
situation changed markedly when people from northwestern and
northern Europe began to travel and migrate to sunnier places in
colonial times, and experience high episodic or sustained loads of
UVR (Jablonski, 2012). Historical migrations of people from north-
ern Europe to far southern Europe, Africa, Australia, and tropical lat-
itudes of the Americas in the last 200 years, along with the increased
popularity of holiday travel, caused dramatic shifts in the nature of
UVR exposure, and increased prevalence of all skin cancers (Latreille
et al., 2009; Rees, 2003; Sturm et al., 2003).
The evolution of skin lightening in northeastern Asia oc-
curred under similar, but not identical, conditions of generally low
and seasonal UVB as those of northwestern Europe (Chaplin &
Jablonski, 2013; Jablonski & Chaplin, 2000). Among the most inter-
esting facts to emerge in the study of the evolution of human skin pig-
mentation evolution is that different suites of genes have influenced
depigmentation in northern Europeans and East Asians. Recent re-
search on the function of the upstream region of KITLG shared by the
common ancestor of all Eurasians shows that the region was under
stronger selective pressure in East Asians, possibly because it con-
ferred depigmentation along with greater resistance to cold (Yang
et al., 2018). This appears to have complemented selection on an
718 | JABLONSKI
OCA2 (oculocutaneous albinism, type 2) variant absent in Europeans
(Yang et al., 2016) and a variant of MFSD12 (Adhikari et al., 2019) to
create a comparable level of depigmentation conducive to cutane-
ous vitamin D production. Thus, depigmentation in the ancestors of
modern western Europeans and East Asians provides an excellent of
convergent evolution (Norton et al., 2007; Yang et al., 2016). Natural
selection has acted upon on different genes and gene variants in re-
sponse to comparable environmental forces to produce comparable
physiological solutions to a problem affecting health and reproduc-
tive success.
East Asians and western Europeans share visibly similar “light”
skin, but their responses to UVR are different. The skin of East
Asians has a higher density of melanosomes and produces more eu-
melanin and pheomelanin in response to UVR exposure than does
the skin or western Europeans (Hennessy, Oh, Diffey, et al., 2005;
Hennessy, Oh, Rees, et al., 2005). Thus, most East Asian people can
tan, whereas many Europeans, especially northern Europeans, can
tan only slightly or not at all. The development and persistence of
tanning abilities are conferred by many genes operating on mela-
nin production, distribution, and breakdown in the skin (Del Bino
et al., 2018) and tanning abilities, like depigmentation, have evolved
multiple times independently in human history under conditions of
seasonally strong UVR (Martínez-­C adenas et al., 2013; Quillen, 2015;
Quillen et al., 2019). The moderate tanning abilities among some
Scandinavians and northern Europeans appear to have been con-
ferred by recent genetic admixture across northern Eurasia (Hanel
& Carlberg, 2020a), but other factors, including altered epistatic in-
teractions among pigmentation genes, may have also contributed.
Following the amelioration of climatic conditions at the end
of the Pleistocene, the development of extensive steppe grass-
lands across much of hinterland Eurasia created a theater for rapid
human population growth associated with animal husbandry, and
relatively rapid, bidirectional east–­
west movements of people
(Allentoft et al., 2015; Damgaard et al., 2018). The results of ge-
nomic, linguistic, and archeological studies show a gradual transi-
tion from Bronze Age pastoralists of West Eurasian ancestry toward
horse-­mounted warrior peoples of increased East Asian ancestry
(Damgaard et al., 2018). The rise and expansion of agriculture from
Anatolia and Iran saw incursions of agriculturalists into southern
and central Europe, increasingly displacing earlier hunter-­gatherer
populations (Skoglund & Mathieson, 2018). The evolution of skin
pigmentation genes and phenotypes of the region reflects these
dramatic movements, and the influence of relatively few genes of
major effect transmitted over long distances by admixture (Ju &
Mathieson, 2020). Of these, the effect of the classic depigmentation
variant of SLC24A5 is greatest, with recent evidence showing that
this variant reached western Europe by admixture from Anatolian
agriculturalists followed by continued positive selection (Ju &
Mathieson, 2020). Recent genomic evidence indicates that some
of the hunter-­gatherer populations of Mesolithic western Europe
and Britain had moved along the coast from the Iberian Peninsula
into northern France, and thence up the Atlantic coast, making
use of marine food resources as they went (Brace et al., 2019). In
this context, the much-­discussed dark or “dark to black” skin re-
constructed for “Cheddar Man,” a Mesolithic inhabitant of Cheddar
Gorge in Somerset, England (Brace et al., 2019), becomes more
clearly understandable and less sensational. Traditional hunter-­
gatherer diets centered around coastal marine sources, hunted
terrestrial game, and foraged plant foods are rich in vitamin D and
make it possible for people to continue to live healthy reproductive
lives while maintaining “dark” and highly tannable skin. Skin depig-
mentation in northwestern Europe occurred gradually, over many
millennia, with the introduction of the classic SLC24A5 variant being
the last and most dramatic step in the process after the introduction
of agriculture.
3.3.1 | Beringia and the Americas
Discussion of the evolution of human skin pigmentation among in-
digenous populations of the Americas follows naturally from that
of northern and northeast Asians because northern Asia was the
area of origin for the earliest peoples entering Beringia and North
America via coastal and overland routes (Goebel et al., 2008;
Moreno-­Mayar et al., 2018; Nielsen et al., 2017). Considerable re-
search and debate over the number, nature, timing, and routing of
these dispersals over the last century have resulted in general con-
sensus that the first humans to disperse into the Americas trave-
led via a coastal route beginning around 15 kya, having genetically
diverged from ancient north Asians beginning around 36 kya with
gene flow continuing until about 25 kya (Moreno-­Mayar et al., 2018;
Nielsen et al., 2017). Details of the population movements are be-
yond the scope of this paper, but a key feature of this dispersal event
was that it involved a prolonged “Beringian standstill” lasting about
15,000 years (Moreno-­Mayar et al., 2018), during which an isolated
population lived under conditions of limited and highly seasonal sun-
light, including virtually no UVB (Hlusko et al., 2018). In the absence
of UVB and cutaneous biosynthesis of vitamin D, the consumption
of vitamin D-­rich foods was necessary for survival and reproduc-
tive success. Because normal human development in utero and in
early postnatal months depends on maternal vitamin D stores, the
evolution of efficient transmission of vitamin D in breastmilk was
critical for infant survival. This need appears to have been met by
positive selection on the human-­
specific EDAR (Ectodysplasin A
receptor) variant V370A associated with mammary ductal branch-
ing, which appears to have amplified the transfer of vitamin D to
infants via mothers’ milk (Hlusko et al., 2018). Evidence for the rapid
spread and positive selection of this variant back into northern Asia
and Scandinavia (Mathieson et al., 2015) suggests that it conferred
an evolutionary advantage to people inhabiting regions in which the
potential for cutaneous production of vitamin D from UVB was se-
verely limited and the vitamin could be derived only from dietary
sources. This example illustrates the importance of understanding
the evolution of skin pigmentation in the broader context of human
evolution and dispersals, and recognizing the nature of life events
most likely to be influenced by natural selection.
JABLONSKI
Skin pigmentation among Inuit people is often discussed, but has
been inadequately measured or genetically characterized. Ancestral
Inuit traversed the Bering Land Bridge in the mid-­Holocene, dispers-
ing into Americas from northern Siberia about 10,000 years after
first coastal dispersal about 15 kya (Nielsen et al., 2017; Skoglund &
Mathieson, 2018). Their constitutive pigmentation is light to mod-
erate, but they have remarkable tanning abilities, as illustrated by
the “tan lines” evident on traditional hunters when their animal skin
parkas are removed (Jablonski, 2012). This is another case of bio-
cultural compromise involving skin pigmentation, dietary vitamin
D, and regulation of vitamin D availability at critical points in the
lifespan. Inuit and their ancestral populations were, traditionally,
hunter-­
gatherers who, depending on location, subsisted primar-
ily on marine mammals, oily fish, and caribou. Living near or above
the Arctic Circle, they experienced markedly seasonal patterns of
daylight and UVR exposure, and negligible UVB; they did, however,
experience seasonally strong direct and reflected UVA (Chaplin &
Jablonski, 2013; Jablonski, 2012; Jablonski & Chaplin, 2000, 2010).
Modest cutaneous production of vitamin D occurs in some popula-
tions, but the physiological significance of this is unclear (Andersen
et al., 2012), In the presence of a traditional diet replete in vitamin
D, and a physiological mechanism to ensure ample vitamin D to rap-
idly developing neonates (Hlusko et al., 2018), there was little se-
lective pressure for further depigmentation; if anything, there may
have been selective pressure to enhance facultative pigmentation.
The fragility of the biocultural compromise here is witnessed by the
high levels of vitamin D deficiency and high prevalence of rickets and
metabolic syndrome in people no longer eating traditional diets year
round (Andersen et al., 2013; El Hayek et al., 2010).
The evolution of skin pigmentation in the Americas has been
studied relatively little, with the exception of a few pioneering stud-
ies in the last decade (Adhikari et al., 2019; Quillen et al., 2012). The
peopling of the Americas from people of northern and northeastern
Asian ancestry at the end of the Pleistocene and during the Holocene
meant that indigenous populations carried novel mixtures of skin
pigmentation genes, which reflect histories of extreme natural se-
lection and population bottlenecks. Study of the evolution of skin
pigmentation in the pre-­Columbian Americas has been made difficult
by the facts that many indigenous populations have not been stud-
ied comprehensively for both their skin pigmentation phenotypes
and genetics, and because most of the populations that have been
studied exhibit evidence of considerable admixture from colonial
European and enslaved sub-­Saharan African populations (Quillen
et al., 2019). In this context, studies in which the effects of genetic
admixture have been “dissected” to reveal novel skin pigmentation
genes have been of key importance (Bonilla et al., 2005; Quillen
et al., 2012). Because of the northern and northeastern Asian origin
of New World populations, the variants of OCA2 and MFSD12 found
in Asian populations are also present among indigenous Americans
(Adhikari et al., 2019; Yang et al., 2016). In addition, other genes not
previously implicated in skin pigmentation, including EGFR (epider-
mal growth factor receptor) and OPRM1 (opioid receptor, mu-­1),
have emerged as important contributors to skin pigmentation among
| 719
indigenous Americans (Quillen et al., 2012, 2019). One of the most
biologically significant facts about the evolution of skin pigmentation
in the Americas is that it has involved selection for genes that confer
enhanced tanning (facultative pigmentation) rather than darker con-
stitutive pigmentation. Most indigenous Americans, including those
that inhabit areas of high and relatively nonseasonal UVR, exhibit
“moderate” skin reflectance values for unexposed skin, but strong
tanning abilities (Jablonski & Chaplin, 2000; Quillen et al., 2019). The
genetic basis of these abilities is almost completely unknown and
warrants considerable study (Quillen et al., 2019). Looking in general
at the evolution of skin pigmentation in the Americas, it is important
to view the range of factors that have contributed to the phenom-
enon: population bottlenecks limiting the pool of potential variant
pigmentation genes, a relatively short history of human habitation,
and the fact that the Late Pleistocene populations dispersing into the
Americas brought with them many means to buffer themselves and
their skin against the exigencies of the environment and seasonal
changes in food availability through sewn clothing, constructed shel-
ters, and food storage technologies (Jablonski, 2012).
3.3.2 | Indian subcontinent
The evolution of skin pigmentation on the Indian subcontinent can
only be understood in light of the complex demographic and social
histories of the region. Because of this complexity and the practical
difficulties of studying living populations on the subcontinent and
obtaining biological samples therefrom, studies of skin pigmentation
diversity and evolution are still in their infancy. The history of dis-
persals and genetic admixture within the subcontinent is complex
and still poorly understood (Dennell & Petraglia, 2012; Majumder &
Basu, 2014; Moorjani et al., 2013). Genetic evidence attests that the
southern coast of the subcontinent received H. sapiens populations
early in the history of dispersal along the coast of the Afro-­Arabian
Peninsula (Majumder & Basu, 2014). Since then, populations dispers-
ing into the subcontinent have followed mostly hinterland routes,
from central and western Asia, primarily via a northwestern corridor
(Damgaard et al., 2018; Majumder & Basu, 2014), southward. Tracing
the fate and routes of these populations has been difficult. The sub-
continent contains people hailing from four distinct language groups
who live in mostly non-­overlapping regions and who reached their
current locations at different times in late prehistory and early his-
tory (Majumder & Basu, 2014). Some populations belong to groups
designated as tribal, while most belong to caste societies. One of the
most distinctive aspects of population structure in the subcontinent
is the pattern of endogamy within groups, including within the hierar-
chically arranged Hindu castes (Majumder & Basu, 2014). Because of
the complex pattern of dispersal and the prevalence of within-­group
marriage, the subcontinent is more accurately described as a genetic
mosaic and, not surprisingly, social factors and population struc-
ture have played a stronger role than natural selection in shaping
skin color diversity across the region (Iliescu et al., 2018; Stokowski
et al., 2007). Two striking findings have emerged from studies of skin
720 | JABLONSKI

F I G U R E 4 Generalized timeline of
main events in the evolution of skin
pigmentation in hinterland Eurasia,
Beringia, and the Americas. (a) In central
Eurasia, beginning around 70 kya,
moderately pigmented and tannable
phenotypes are favored under continued
relaxation of selection on MC1R and
positive selection of a KITLG producing
lighter skin. After the introduction of
agriculture and animal husbandry around
10 kya, increasing population admixture
with diverse skin pigmentation gene
assemblages producing moderately
pigmented, tannable skin. (b) In
northern and northwestern Europe, the
introduction of the SLC24A5 variant
from Anatolian agriculturalists around
6 kya is followed by positive selection
on the variant; this is favored strongly
in relatively isolated populations living
under the low and seasonal UVB
conditions of northernmost Europe.
(c) In East Asia, intensified selection
on the KITLG variant and other loci
produces further depigmentation. (d)
In Beringia, hunter-­gather populations
experiencing the “Beringian standstill”
from about 25–­15 kya undergo selection
for the EDAR variant favoring enhanced
mammary transmission of vitamin D, while
maintaining or enhancing their tanning
abilities through skin pigmentation genes
not yet identified. (e) In the Americas,
technologically sophisticated hunter-­
gathers disperse southward, carrying
genes capable of producing strong
tans under high UVB conditions. Dark
constitutive pigmentation does not
evolve, probably because of the absence
of appropriate gene variants and the many
cultural buffering mechanisms—­including
food storage—­used by dispersing
populations to mitigate the effects
of different regimes of UVB and food
availability
JABLONSKI
pigmentation diversity in the subcontinent to date. The first is that
while the range of skin pigmentation phenotypes across the breadth
and length of the subcontinent is vast (Jablonski & Chaplin, 2000),
the pattern is not strongly correlated to strength of UVR (Iliescu
et al., 2018). The second is that the classic skin lightening variant of
SLC24A5 is present in high frequencies in most populations but is
not expressed (Basu Mallick et al., 2013; Iliescu et al., 2018). Thus,
epistasis has affected the expression of the SLC24A5 in such a way as
to prevent the allele's ability to lower eumelanin production (Quillen
et al., 2019). The reasons behind this can be traced potentially to
the specific and distinct genetic backgrounds, including social selec-
tion and sexual selection, that contributed to the unique reproduc-
tive structure of the Indian populations, but the relative importance
of these phenomena is not clear at present. This result should be
seen as a useful cautionary tale for forensic reconstruction of skin
pigmentation, viz., specific genotypes are not associated reliably
with specific skin color phenotypes (Iliescu et al., 2018; Quillen
et al., 2019).
A hypothetical skin pigmentation timeline for the major regions
of Eurasia and the Americas is presented in Figure 4.
4 | S E X UA L D I M O R PH I S M A N D
S E X UA L S E LEC TI O N I N H U M A N S K I N
PI G M E NTATI O N
One of the most interesting and still not fully understood aspects
of human skin pigmentation evolution is the consistent pattern of
sexual dimorphism observed: In most indigenous groups whose
skin phenotypes have been quantified using reflectometry, female
pigmentation is lighter than male, sometimes to a striking degree
(Byard, 1981; Jablonski & Chaplin, 2000; Roberts & Kahlon, 1972).
This aspect of human skin pigmentation garnered early attention
from natural historians including Charles Darwin and has been the
focus of considerable attention since. Darwin believed that differ-
ences in human skin color between “races” were caused, not by
natural selection, but by sexual selection; in other words, that skin
color had been primarily and systematically influenced by deliber-
ate choice of mates (Aoki, 2002; Darwin, 1871). The belief that skin
color is an important determinant of human mate choice became
fixed in the literature and public imagination in the late 20th cen-
tury (Aoki, 2002; Diamond, 1991; Robins, 1991; Van den Berghe &
Frost, 1986). Further study has revealed, however, that sexual selec-
tion is not as dominant and omnipresent force as some have con-
ceived (Jablonski & Chaplin, 2000; Madrigal & Kelly, 2007). Sexual
dimorphism in human skin pigmentation is real, but its expression
varies greatly, with some populations exhibiting marked levels and
others very little (Jablonski & Chaplin, 2000; Norton et al., 2006).
The primary cause of the difference between the sexes may be the
importance of increased cutaneous vitamin D production poten-
tial, and therefore lighter skin, in the skin of reproductively aged
women, in order to facilitate absorption and redistribution of dietary
calcium to the developing fetus and nursing neonate (Jablonski &
| 721
Chaplin, 2000). Satisfactory tests of this hypothesis in populations
with varying levels of skin color sexual dimorphism have not yet been
undertaken. What seems likely is that sexual selection has prob-
ably played a secondary and complementary role in the evolution
of skin pigmentation, primarily through manipulating levels of exist-
ing sexual dimorphism (Jablonski, 2012; Jablonski & Chaplin, 2000;
Quillen et al., 2019). This apparent directional preference for women
with lighter skin appears to be particularly strongly marked in Island
Melanesia, Japan, and among certain populations in India where
cultural preferences for lighter females are strongly culturally rein-
forced (Iliescu et al., 2018; Jablonski, 2012; Norton et al., 2006). It
would be surprising if uniform, global patterns of sexual selection
applied because these would rely on uniform preferences (Quillen
et al., 2019). Human skin pigmentation is the product of many cul-
tural forces, including culturally determined mating preferences
and sexual selection, which have had potent local effects modify-
ing the influence of UVR-­determined directional selection (Quillen
et al., 2019).
5 | CO N C LU S I O N S
The evolution of human skin pigmentation has never been a sim-
ple deterministic process, but it has been influenced potently by
environmental UVR and its effects on vitamin availability. Humans
evolved under the sun. The early evolution of the genus Homo oc-
curred in equatorial Africa under conditions of high UVA and high
UVB. Under these conditions, natural selection favored the evolu-
tion of enhanced sweating abilities and enhanced permanent eu-
melanin pigmentation, concomitant with the loss of most body hair.
Enhanced eumelanin constitutive pigmentation provided protection
against folate degradation and damage to DNA while still permit-
ting cutaneous vitamin D production. Dark skin in early H. sapiens
was made possible by elimination of variation at the MC1R locus and
positive selection at the MFSD12, DDB1, and probably other loci.
Populations of H. sapiens in equatorial Africa underwent further posi-
tive selection for very dark, eumelanin-­rich pigmentation. Habitation
of southern Africa, with its somewhat lower and more seasonal UVR
regimes, involved relaxation of purifying selection on MC1R. Further
depigmentation in southern African hunter-­gatherers occurred
under the influence of the classic variant of SLC24A5, which en-
tered Africa through the Afro-­Arabian Peninsula and rapidly spread
southward. Dispersal of H. sapiens into Eurasia began around 70 kya
and involved both rapid coastal and hinterland routes. Dispersing
populations were small, and population bottlenecks affected the na-
ture of pigmentation gene variants they carried. Equipped with so-
phisticated food procurement technologies, but not sewn clothing,
agriculture or animal husbandry, people modified their diets and life-
styles according to local resources and conditions. Coastal dispersal
routes in South and Southeast Asia and Europe afforded people the
opportunity to easily harvest fish and other vitamin D-­rich foods,
mitigating selective pressure for depigmentation to facilitate cutane-
ous production of vitamin D. Further darkening of skin pigmentation
722 | JABLONSKI
occurred, involving intensified selection on MFSD12, DDB1, and
probably some variants carried by introgressing Denisovans, in
predominantly coastal populations dispersing into equatorial island
Southeast Asia, Melanesia, and Australia. Habitation of non-­tropical
hinterland Eurasia with more highly seasonal UVB regimes saw the
evolution of lighter constitutive pigmentation under the influence
of positive selection for a variant of KITLG, which was then shared
by populations dispersing into northern and western Europe, and
northeastern Asia. Trends toward loss of constitutive eumelanin pig-
mentation and variable tanning abilities occurred at different times
in these northward diverging populations and involved different
suites of pigmentation genes. Prior to the introduction of agriculture
and the origin of the classic variant of SLC24A5, hinterland Eurasians
were moderately pigmented, had some or considerable tanning abili-
ties, probably conferred by varying configurations of pigmentation
genes, and pursued outdoor hunter-­gatherer lifestyles. The evolu-
tion of extremely depigmented skin was a recent novelty in human
evolution, occurring in northern and northwestern Europe after the
introduction of the SLC25A5 variant as the result of admixture with
agriculturalists. Skin containing little eumelanin and lacking tanning
abilities evolved only in relatively isolated populations living under
the lowest and most highly seasonal UVB regimes with very limited
opportunities for cutaneous vitamin D synthesis. The extremes of
human skin pigmentation evolved under extremes of environmen-
tal UVB and were mitigated by vitamin D-­rich diets. When genetic
variants were available that enhanced reproductive success, they
underwent rapid positive selection, especially in extreme solar en-
vironments, and quickly brought about changes in skin pigmentation
phenotypes. Thus, natural selection for dark pigmentation under
high UVR conditions and for lighter skin capable of tanning under
lower and more seasonal UVR has been the dominant influence, but
skin pigmentation has been modified increasingly by population ge-
netic influences on the genetic composition of dispersing popula-
tions and by cultural processes, which have mitigated the expression
of pigmentation genes and modified the impact of the environment
on the human body. In recent millennia, human skin pigmentation
has been influenced increasingly by culturally reinforced processes
including, in some places, mating practices and sexual selection.
There remains a pressing need for more transdisciplinary re-
search on the evolution of human pigmentation diversity. These
need to include functional genomic studies aimed at further eluci-
dating the mechanisms of tanning, as well as studies aimed at un-
derstanding the effects of genetic admixture and epistasis, and
epigenetic influences on melanin production. We also need to bet-
ter understand how various combinations of melanin pigments and
the physical packaging of melanosomes contribute to the subtly
different colors observed in human skin. It has long been appreci-
ated that different forms of melanin in the skin—­DHICA-­eumelanin,
DHI-­eumelanin, and pheomelanin—­contribute to the chromatic vari-
ation in skin color phenotypes (Alaluf et al., ,2001, 2002), but much
still remains to be clarified about how the different melanin types
and, possibly also, the different sizes and physical arrangements of
melanosomes impart subtly different colors to skin. Lastly, study of
the contributions of systematic cultural or sexual selection to pro-
ducing directional evolution of skin pigmentation in recent history
also requires further, thoughtful study, but such work must be pur-
sued with careful attention to potential investigator bias, based on
individual or cultural preoccupations. The evolution of human skin
pigmentation has been a complex and contingent process for hun-
dreds of thousands of years. This should not discourage us, because
we now have the tools to understand the many genetic and cultural
factors that have contributed to it.
AC K N OW L E D G E M E N T S
I am grateful to the editors of the special issue of Pigment Cell and
Melanoma Research for their invitation to contribute. Shosuke Ito is
thanked especially for supervising the review of the manuscript. The
ideas in this paper were developed over the course of many years,
and I am grateful especially to George Chaplin for lengthy and prob-
ing discussions of all of these over the last 25 years. In recent years,
I have benefited from clarifying discussions with Roger Bouillon, Tim
Caro, Keith Cheng, Brenna Henn, Michael Holick, Mircea Iliescu,
Larry Kenney, Beatriz Marcheco-­Teruel, Heather Norton, Richard
Prum, Ellen Quillen, Mark Shriver, Rick Sturm, Mark Thomas, Sarah
Tishkoff, Richard Weller, Tony Wolf, and Antony Young. I thank my
research assistant, Tess Wilson, for her continuing excellence and
support in maintaining bibliographic materials, producing figures,
and providing all-­around logistical support for my research.
ORCID
Nina G. Jablonski https://orcid.org/0000-0001-7644-874X
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 The evolution of skin pigmentation-associated
variation in West Eurasia
Dan Jua,1 and Iain Mathiesona,1
a
Department of Genetics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104
Edited by Anne C. Stone, Arizona State University, Tempe, AZ, and approved November 4, 2020 (received for review May 11, 2020)
Skin pigmentation is a classic example of a polygenic trait that has
experienced directional selection in humans. Genome-wide associ-
ation studies have identified well over a hundred pigmentation-
associated loci, and genomic scans in present-day and ancient pop-
ulations have identified selective sweeps for a small number of
light pigmentation-associated alleles in Europeans. It is unclear
whether selection has operated on all of the genetic variation as-
sociated with skin pigmentation as opposed to just a small number
of large-effect variants. Here, we address this question using an-
cient DNA from 1,158 individuals from West Eurasia covering a
period of 40,000 y combined with genome-wide association sum-
mary statistics from the UK Biobank. We find a robust signal of
directional selection in ancient West Eurasians on 170 skin
pigmentation-associated variants ascertained in the UK Biobank.
However, we also show that this signal is driven by a limited num-
ber of large-effect variants. Consistent with this observation, we
find that a polygenic selection test in present-day populations fails
to detect selection with the full set of variants. Our data allow us
to disentangle the effects of admixture and selection. Most nota-
bly, a large-effect variant at SLC24A5 was introduced to Western
Europe by migrations of Neolithic farming populations but contin-
ued to be under selection post-admixture. This study shows that
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the response to selection for light skin pigmentation in West Eur-
asia was driven by a relatively small proportion of the variants
that are associated with present-day phenotypic variation.
skin pigmentation | polygenic selection | complex traits | evolution |
ancient DNA
S kin pigmentation exhibits a gradient of variation across hu-
man populations that tracks with latitude (1). This gradient is
thought to reflect selection for lighter skin pigmentation at higher
latitudes, as lower UVB exposure reduces vitamin D biosynthesis,
which affects calcium homeostasis and immunity (2–4). Studies of
present-day and ancient populations have revealed signatures of
selection at skin pigmentation loci (5–9), and single-nucleotide
polymorphisms (SNPs) associated with light skin pigmentation at
some of these genes exhibit a signal of polygenic selection in
Western Eurasians (10). However, this observation, the only docu-
mented signal of polygenic selection for skin pigmentation, is based
on just four loci (SLC24A5, SLC45A2, TYR, and APBA2/OCA2).
Therefore, while the existence of selective sweeps at a handful
of skin pigmentation loci is well established, the evidence for
polygenic selection—a coordinated shift in allele frequencies
across many trait-associated variants (11)—is less clear. Re-
cently, genome-wide association studies (GWAS) of larger sam-
ples and more diverse populations (12–15) have emphasized the
polygenic architecture of skin pigmentation. This raises the
question of whether selection on skin pigmentation acted on all of
this variation or was indeed driven by selective sweeps at a rela-
tively small number of loci (11).
The impact of demographic transitions on the evolution of
skin pigmentation also remains an open question. The Holocene
history (∼12,000 y before present [BP]) of Europe was marked by
waves of migration and admixture between three highly diverged
populations: hunter-gatherers, Early Farmers, and Steppe an-
cestry populations (16, 17). Skin pigmentation-associated loci
PNAS 2021 Vol. 118 No. 1 e2009227118
may have been selected independently or in parallel in one or
more of these source populations or may instead only have been
selected after admixture. The impact of ancient shifts in ancestry
is difficult to resolve using present-day data, but using ancient
DNA, we can separate the effects of ancestry and selection and
identify which loci were selected in which populations.
Here, we use ancient DNA to track the evolution of loci that
are associated with skin pigmentation in present-day Europeans.
Although we cannot make predictions about the phenotypes of
ancient individuals or populations, we can assess the extent to
which they carried the same light pigmentation alleles that are
present today. This allows us to identify which pigmentation-
associated variants have changed in frequency due to positive
selection and the timing of these selective events. We present a
systematic survey of the evolution of European skin pigmentation-
EVOLUTION
associated variation, tracking over a hundred loci over 40,000 y of
human history—almost the entire range of modern human occu-
pation of Europe.
Results
Skin Pigmentation-Associated SNPs Show a Signal of Positive Selection
in Ancient West Eurasians. We obtained skin pigmentation-associated
SNPs from the UK Biobank GWAS for skin color released by the
Neale Lab (14). We analyzed the evolution of these variants using
two datasets of publicly available ancient DNA (Fig. 1 A–C and SI
Appendix, Fig. S1). The first (“capture-shotgun”) consists of data
from 1,158 individuals dating from 45,000–715 y BP genotyped at
∼1.2 million variants (7, 16, 18–52). The second (“shotgun”) is a
Significance
Some of the genes responsible for the evolution of light skin
pigmentation in Europeans show signals of positive selection
in present-day populations. Recently, genome-wide association
studies have highlighted the highly polygenic nature of skin
pigmentation. It is unclear whether selection has operated on
all of these genetic variants or just a subset. By studying vari-
ation in over a thousand ancient genomes from West Eurasia
covering 40,000 y, we are able to study both the aggregate
behavior of pigmentation-associated variants and the evolu-
tionary history of individual variants. We find that the evolu-
tion of light skin pigmentation in Europeans was driven by
frequency changes in a relatively small fraction of the genetic
variants that are associated with variation in the trait today.
Author contributions: D.J. and I.M. designed research; D.J. and I.M. performed research;
I.M. contributed new reagents/analytic tools; D.J. analyzed data; and D.J. and I.M. wrote
the paper.
The authors declare no competing interest.
This article is a PNAS Direct Submission.
This open access article is distributed under Creative Commons Attribution License 4.0
(CC BY).
1
To whom correspondence may be addressed. Email: danju@pennmedicine.upenn.edu or
mathi@pennmedicine.upenn.edu.
This article contains supporting information online at https://www.pnas.org/lookup/suppl/
doi:10.1073/pnas.2009227118/-/DCSupplemental.
Published December 21, 2020.
https://doi.org/10.1073/pnas.2009227118 | 1 of 8
 Shotgun (40-1 kyBP) Capture-shotgun (40-1 kyBP) Capture-shotgun (15-1 kyBP)

A B C
1.0 1.0 1.0 Population
EF
HG
AFR SP
Ust Ishim Anzick Ust Ishim Mota
MA−1
EAS AFR
Tianyuan Mota Anzick
SAS Tianyuan MA−1 EAS
UK Biobank

EUR SAS

Score
Score

Score
0.5 0.5 EUR 0.5

242 SNPs 170 SNPs 170 SNPs

n = 248 n = 1158 n = 1079
p=2.67x10-3 p<1x10-4 p<1x10-4
0.0 0.0 0.0

40000 30000 20000 10000 0 40000 30000 20000 10000 0 10000 5000
Date (years BP) Date (years BP) Date (years BP)

D E F
MA−1
1.0 1.0 1.0 Population
EF
HG
SP
Ust Ishim
AFR
Manually curated

Tianyuan
Mota
Anzick
Tianyuan MA−1 Anzick AFR
Mota EAS
Score

Score

Score
Ust Ishim EAS
SAS
0.5 SAS 0.5 0.5

18 SNPs EUR 10 SNPs EUR

10 SNPs
n = 225 n = 1093 n = 1018
p=0.17 p<1x10-4 p<1x10-4
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0.0 0.0 0.0

40000 30000 20000 10000 0 40000 30000 20000 10000 0 10000 5000
Date (years BP) Date (years BP) Date (years BP)

Fig. 1. Genetic scores for skin pigmentation over time. The solid lines indicate fitted mean scores with gray 95% confidence intervals. Weighted scores based
on UK Biobank SNPs and unweighted scores based on manually curated SNPs for samples in the (A and D) shotgun dataset, (B and E) capture-shotgun dataset,
and (C and F) capture-shotgun dataset dated within the past 15,000 y. Scores of labeled samples are plotted but not included in regressions. Averages for 1000
Genomes superpopulations are plotted at time 0. Area of points scale with number of SNPs genotyped in the individual.

subset of 248 individuals with genome-wide shotgun sequence data
(16, 18–25, 28, 32, 35, 38, 40–44, 47, 49, 51, 53–60). This smaller
dataset allows us to capture variants that may not be well tagged by
the genotyped SNPs in the capture-shotgun dataset. Weighting
variants by their GWAS-estimated effect sizes, we show that the
polygenic score—the weighted proportion of dark pigmentation
alleles—decreased significantly over the past 40,000 y (Fig. 1 A and
B, P < 1 × 10−4 based on a genomic null distribution and accounting
for changes in ancestry; SI Appendix, Fig. S2). We recapitulate this
significant decrease using a different set of UK Biobank GWAS
summary statistics calculated using linear mixed models (61) (SI
Appendix, Fig. S3) and identifying independent signals based on
predefined approximately independent linkage disequilibrium (LD)
blocks (62) (SI Appendix, Fig. S4), rather than LD clumping. The
genetic scores of the oldest individuals in the dataset fall within the
range of present-day West African populations, showing that Early
Upper Paleolithic (∼50–20,000 y BP) individuals, such as Ust’Ishim,
carried few of the light skin pigmentation alleles that are common in
present-day Europe.
Over the past 15,000 y, which covers most of the data, the
polygenic score decreased (P < 1 × 10−4) at a similar rate to the
estimated rate over the 40,000-y period (5.27 × 10−5 per year vs.
3.47 × 10−5 per year; P = 0.04). Stratifying individuals by ancestry
(hunter-gatherer, Early Farmer, and Steppe) using ADMIX-
TURE revealed baseline differences in genetic score across
groups (Fig. 1C). Mesolithic hunter-gatherers carried fewer light
pigmentation alleles than Early Farmer or Steppe ancestry pop-
ulations. This difference (0.091 score units) was similar to the
difference between Mesolithic hunter-gatherers and Early Upper
Paleolithic populations (0.097 score units). We tested for evidence
of change in score over time within groups while controlling for
changes in ancestry, finding no significant change within hunter-
gatherer or Early Farmer populations (P = 0.32; P = 0.29). Steppe
ancestry populations, however, exhibited a significant decrease
(P = 0.007), with a steeper slope than that of Early Farmer or
hunter-gatherer (P = 0.004 and P = 0.08, respectively).
While the UK Biobank is well powered to detect variation that
is segregating in present-day Europeans, it may not detect vari-
ants that are no longer segregating. We therefore manually cu-
rated a separate list of 18 SNPs identified as associated with skin
pigmentation from seven studies of populations representing
diverse ancestries (Dataset S1E). Using this set of SNPs to
generate unweighted scores, we observed similar results as with
the UK Biobank ascertained SNPs (Fig. 1 D–F). We find a sig-
nificant decrease in genetic score over both 40,000 and 15,000 y
in the capture-shotgun dataset (P < 1 × 10−4 for both), although
the decrease in the shotgun dataset was not significant (P =
0.17), probably reflecting a lack of power from the small number
of ancient samples and SNPs.

2 of 8 | PNAS Ju and Mathieson

https://doi.org/10.1073/pnas.2009227118 The evolution of skin pigmentation-associated variation in West Eurasia
 Ancestry and Selection Have Different Effects on the Histories of Each
Variant. Next, we investigated the evolution of each variant in-
dependently. We separated the effects of ancestry and selection
in the data by regressing presence or absence of the alternate
allele for each pigmentation-associated SNP on both date and
ancestry, inferred using principal component analysis (PCA)
(63). Significant effects of ancestry on frequency can be inter-
preted as changes in allele frequency that can be explained by
changes in ancestry. Significant changes in frequency over time,
after accounting for ancestry, can be interpreted as evidence for
selection occurring during the analyzed time period.
The SNP rs16891982 at the SLC45A2 locus shows the stron-
gest evidence for selection across analyses (Fig. 2), but we note
this does not necessarily imply positive selection over the entire
time transect. We observed little evidence for demographic tran-
sitions in driving the increase in light allele frequency over time for
this SNP. In addition, we found robust signals of selection for the
light allele of rs1126809 near TYR, rs12913832 and rs1635168 near
HERC2, and rs7109255 near GRM5 (Fig. 2 B and C). In some
cases—for example at rs6120849 near EDEM2 and rs7870409
near RALGPS1—light alleles decreased in frequency more than
can be explained by changes in ancestry (Fig. 2 B and C). The dark
A B
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D E
Fig. 2. Plot of P values for ancestry and time for individual skin pigmentation SNPs. The dashed lines indicate fifth percentile of P values. UK Biobank SNPs
using the shotgun dataset are plotted in A and capture-shotgun dataset in B and C. Manually curated SNPs using the shotgun dataset are plotted in D and
capture-shotgun dataset in E and F. SNPs are colored according to whether the light allele is increasing over time (red) or decreasing (blue), with saturation
determined by the magnitude of change.
Ju and Mathieson
The evolution of skin pigmentation-associated variation in West Eurasia
allele at rs6120849 (EDEM2) is also significantly associated with
several other traits including increased sitting and standing height
in the UK Biobank, increased blood creatinine, and increased
heel bone mineral density (64). The dark allele of rs7870409
(RALGPS1) is associated with increased arm and trunk fat per-
centage (65). These observations raise the possibility of pleiotropic
effects constraining the evolution of these loci or of spurious
pleiotropy due to uncorrected population stratification in the
GWAS.
On the other hand, the changes in frequencies of several vari-
ants appear to have been driven largely by changes in ancestry. For
example, rs4778123 near OCA2, rs2153271 near BNC2, and
rs3758833 near CTSC show evidence that their changes in fre-
quency were consistent with changes in genome-wide ancestry
(Fig. 2 B and C). At SLC24A5, rs2675345 shows evidence of
change with both ancestry and time, suggesting that even after the
spread of the light allele from Anatolia into Western Europe in
the Neolithic (7) selection continued to occur post-admixture.
Again, not all of these cases involve an increase in the frequency of
the light pigmentation allele over time. The light allele of
rs4778123 (OCA2) was at high frequency in hunter-gatherers but
lower in later populations (SI Appendix, Fig. S6B). From the
EVOLUTION
C
F
PNAS | 3 of 8
https://doi.org/10.1073/pnas.2009227118
 manually curated set of SNPs (Fig. 2 D–F), rs12203592 near IRF4
also displays a marked effect of ancestry with higher light allele
frequency in hunter-gatherers (SI Appendix, Fig. S6E). While
rs12203592 was not present in the UK Biobank summary statistics,
another SNP at the IRF4 locus, rs3778607, was present but with a
smaller ancestry effect (SI Appendix, Fig. S7).
Lack of PBS Selection Signal across Pigmentation SNPs in Source
Populations of Europeans. As we described in the previous sec-
tion, the frequencies of several skin pigmentation SNPs appear
to have been driven by changes in ancestry. This observation
suggests that their frequencies have diverged between the Eu-
ropean source populations and then subsequently been driven by
admixture. Frequency differences across the source populations
might reflect the action of either genetic drift or selection. To
examine this question, we looked for evidence of selection at skin
pigmentation SNPs in each ancestral population using the pop-
ulation branch statistic (PBS) test (66). We ran ADMIXTURE
with K = 3 on the capture-shotgun dataset (SI Appendix, Fig. S8)
and used the inferred source population allele frequencies to
calculate PBS for each source in 20-SNP nonoverlapping win-
dows, with the 1000 Genomes CHB (Han Chinese) and YRI
(Yoruba) populations as outgroups.
Few of the skin pigmentation loci show extreme PBS values
(Fig. 3), and even fewer show evidence of divergent frequencies
across ancient groups (SI Appendix, Fig. S10). On the Early
Farmer and Steppe ancestry branches, but not the hunter-
gatherer branch, the SLC24A5 locus exhibited the strongest
signal (Fig. 3 B and C), indicating selection at the locus in both
Early Farmer and Steppe source populations. The RABGAP1
locus also exhibited an elevated signal of selection in Early
Farmer and Steppe, which remained when using a 40-SNP win-
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dow size (SI Appendix, Fig. S11 B and C). In all three ancestry
groups, we observed an elevated PBS at the OCA2 locus, with
the most extreme value on the hunter-gatherer branch. However,
unlike the SLC24A5 variant, neither of these SNPs (rs644490
and rs9920172) showed a substantial effect of ancestry from the
individual SNP regression analysis. As a whole, the PBS values of
the 170 skin pigmentation SNPs do not significantly deviate from
the genome-wide distribution of PBS for any of the ancient
source populations (SI Appendix, Fig. S12).
Signals of Selection Are Restricted to the SNPs with the Largest Effect
Sizes. Since our results suggest that pigmentation-associated
variants exhibit different changes in allele frequency, we fur-
ther examined the signal of selection found in Fig. 1. In general,
SNPs with larger GWAS effect sizes changed more in frequency
Fig. 3. Joint PBS distributions for 20-SNP windows across the genome for GBR-CHB-YRI on the x axis and X-CHB-YRI on the y axis, with X being (A) hunter-
gatherer, (B) Early Farmer, and (C) Steppe. The boxes represent windows centered around UK Biobank skin pigmentation SNPs and are colored redder according
to how much higher the light allele frequency is in X compared to YRI. Nearest genes are labeled. The orange and red lines represent the top 1 and 0.1 percentiles.
4 of 8 | PNAS
https://doi.org/10.1073/pnas.2009227118
over time than those with small effect sizes (P = 2.8 × 10−18;
adjusted R2 = 0.36) (SI Appendix, Fig. S13A). To understand the
contribution of these large GWAS effect size SNPs, we itera-
tively removed SNPs from the polygenic score calculations (Fig.
4A). Removing the SNP rs16891982 at SLC45A2, we still found a
significant decrease of score over time (P < 1 × 10−4), but the
estimated rate of change (βtime) decreased by 39%. Removing
the top two SNPs at the SLC45A2 and SLC24A5 locus further
attenuated the signal with a decrease in βtime of 58% (P = 3.96 ×
10−4). We removed the five SNPs with the largest GWAS effect
sizes and found the effect of time attenuated (P = 0.026) with a
decrease in βtime of 78%. Removing the top 10 SNPs attenuated
the signal even more (P = 0.050) with an 81% decrease and
removal of 15 or 20 SNPs abolished the signal (P = 0.147,
P = 0.52).
Using present-day populations from the 1000 Genomes Proj-
ect (67), we tested for polygenic selection with the Qx statistic
(10), which measures overdispersion of trait-associated SNPs
relative to the genome-wide expectation. We performed the test
with different numbers of SNPs ordered by largest to smallest
absolute GWAS effect size (Fig. 4B). Using all 170 SNPs in the
test, we failed to observe a signal of polygenic selection (P =
0.27). However, when we restricted to the top five largest GWAS
effect size SNPs, we found a significant signal (P < 2.5 × 10−7).
We observed significant Qx statistics at P < 0.05 up to the top 30
SNPs, but it appears this signal is driven by the top SNPs of
largest effect. After removing the top 5 SNPs and testing the
remaining 25 top SNPs, the signal of polygenic selection dis-
appeared (P = 0.59). Examining the predicted variance explained
per SNP (Fig. 4C and SI Appendix, Fig. S14), we predict that, of
the UK Biobank SNPs, two SNPs at SLC45A2 and SLC24A5
make the largest contribution to the difference between West
African and European populations. Most UK Biobank skin
pigmentation-associated SNPs are predicted to contribute rela-
tively little to the between-population variance.
Discussion
Large whole-genome ancient DNA datasets have, in the past few
years, allowed us to track the evolution of variation associated
with both simple and complex traits (68, 69). The majority of
samples are from Western Eurasia, and we focus on that region,
noting that a parallel process of selection for light skin pig-
mentation has acted in East Asian populations (9, 70). In West
Eurasia, selection for skin pigmentation appears to have been
dominated by a small number of selective sweeps at large-effect
variants. There are a number of possible explanations for this.
Many of the variants detected by the UK Biobank GWAS may
Ju and Mathieson
The evolution of skin pigmentation-associated variation in West Eurasia
 A B
Fig. 4. (A) Regressions of genetic score based on UK Biobank SNPs using the capture-shotgun dataset over date, with scores using all SNPs and iteratively
removing top GWAS effect size SNPs. (B) Qx empirical −log10(P values) using UK Biobank skin pigmentation-associated variants with all 1000 Genomes
populations. Different numbers of SNPs were used to calculate Qx, which were ordered by GWAS-estimated effect size. (C) Variance explained by UK Biobank
SNPs with nearest gene labeled between UK and YRI populations (y axis) and within the UK population (x axis).
have such small effects that they were effectively neutral or ex-
perienced such weak selection that we cannot detect it. Other
variants may not have responded to selection because of pleio-
tropic constraint. The five SNPs with the largest effect sizes in
UK Biobank do have some have small but significant associa-
tions with anthropometric phenotypes (for example, rs1805007
at MC1R with standing height), but by far their most significant
associations are with hair color, facial aging, tanning, melanoma
risk, and other phenotypes that are likely related to pigmentation
(64). Finally, GWAS effect size estimates in European pop-
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ulations today may not reflect the impact these variants had in
the past due to epistatic or gene–environment interactions. This
would not affect the individual SNP time series and PBS analyses
since they do not rely on effect sizes as weights but would reduce
power for the weighted polygenic score and Qx analyses.
Our analyses centered on 170 skin pigmentation-associated
SNPs that are present on the 1240K capture array. To check
for possible bias introduced by the capture array, we checked
how many of the 242 SNPs used in the shotgun analysis were well
tagged by the capture array. Of the 242 SNPs, 33 are shared, a
further 67 are tagged at r2 ≥ 0.8, and a further 19 SNPs are
tagged at r2 ≥ 0.5. However, while the 142 SNPs that are not
tagged at r2 ≥ 0.8 by the capture array do contain some moderate
effect size SNPs, they do not contribute to the selection signal
(P = 0.31) (SI Appendix, Fig. S15D). Therefore, although the
capture array does not capture all of the variation contributing to
present-day skin pigmentation variation, it does capture the vast
majority of the variation that contributed to the evolution of the
phenotype, justifying the use of the capture-shotgun dataset for
detailed investigation of the evolutionary trends.
We find little evidence of parallel selection on independent
haplotypes at skin pigmentation loci, suggesting that that dif-
ferences in allele frequency across ancestry groups were mostly
due to genetic drift. One exception is that the light allele at
SLC24A5 was nearly fixed in both Early Farmer and Steppe
ancestry populations due to selection. However, even for this
variant we observe a signal of ongoing selection in our data even
after admixture with hunter-gatherers, indicating continued se-
lection after admixture. This is analogous to the rapid selection
at the same locus for the light allele introduced via admixture
into the KhoeSan, who now occupy southern Africa (12, 71).
We are also able to test previous claims about selection on
particular pigmentation genes. We find no evidence of positive
selection in Europeans at the MC1R locus in contrast to previous
reports (72, 73). Among UK Biobank SNPs, rs1805007 near
Ju and Mathieson
The evolution of skin pigmentation-associated variation in West Eurasia
C
MC1R explains a relatively large amount of variation within the
UK but is predicted to explain relatively little of the variation
between Europe and West Africa (Fig. 4C). The TYRP1 locus
EVOLUTION
has been previously identified as a target of selection in Euro-
peans (5, 8, 9, 74, 75), although some studies (76, 77) have
questioned this finding. Our analysis shows some support for
selection at this locus, with a 20-SNP window centered around
rs1325132 being in the top 1.1% of genome-wide PBS windows
on the hunter-gatherer lineage. Some studies (76, 78) detect a
signal of recent selection in Europeans at the KITLG SNP
rs12821256 that is functionally associated with blond hair color
(79). We find no evidence of selection on the West Eurasian
lineage at this SNP in our time series analysis, but this may reflect
a lack of power to detect a relatively small change in frequency.
Finally, at the OCA2 locus, we recapitulate an observation of in-
dependent selection in Europeans and East Asians (70, 80) (SI
Appendix, Fig. S9). In the ancient populations, we found an ele-
vated PBS signal around rs9920172 that was most prominent in
hunter-gatherers but also elevated in Early Farmer and Steppe
ancestry populations (Fig. 3), suggesting a relatively early episode
of selection in West Eurasians.
Relatively dark skin pigmentation in Early Upper Paleolithic
Europe would be consistent with those populations being relatively
poorly adapted to high-latitude conditions as a result of having
recently migrated from lower latitudes. On the other hand, although
we have shown that these populations carried few of the light pig-
mentation alleles that are segregating in present-day Europe, they
may have carried different alleles that we cannot now detect. As an
extreme example, Neanderthals and the Altai Denisovan individual
show genetic scores that are in a similar range to Early Upper
Paleolithic individuals (SI Appendix, Table S1), but it is highly
plausible that these populations, who lived at high latitudes for
hundreds of thousands of years, would have adapted independently
to low UV levels. For this reason, we cannot confidently make
statements about the skin pigmentation of ancient populations.
Our study focused, for reasons of data availability, on the
history of skin pigmentation evolution in West Eurasia. How-
ever, there is strong evidence that a parallel trend of adaptation
to low UVB conditions occurred in East Asia (15, 70, 81, 82).
Less is known about the loci that have been under selection in
East Asia, aside from some variants at OCA2 (80–82). Similarly,
multiple studies have documented selection for both lighter and
darker skin pigmentation in parts of Africa (12, 13, 83). Future
work should test whether the process of adaptation in other parts
of the world was similar to that in Europe. The lack of known
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 skin pigmentation loci in scans of positive selection in East Asian
populations (84, 85) raises the possibility that selection may have
been more polygenic in East Asians than in Europeans. Finally,
the evidence for polygenic directional selection on other complex
traits in humans is inconclusive (86, 87). We suggest that detailed
studies of other phenotypes using ancient DNA can be helpful at
more generally identifying the types of processes that are im-
portant in human evolution.
Methods
Capture-Shotgun Ancient DNA Dataset. We downloaded version 37.2 of the
publicly available datasets of 2,107 ancient and 5,637 present-day (Human
Origins dataset) samples from the Reich Lab website: https://reich.hms.harvard.
edu/downloadable-genotypes-present-day-and-ancient-dna-data-compiled-
published-papers. Ancient individuals were treated as pseudohaploid (i.e.,
carry only one allele) because most samples are low coverage. For many of
these samples, 1,233,013 sites were genotyped using an in-solution capture
method (88), while those with whole-genome shotgun sequence data were
genotyped at the same set of sites. For our regression models, we included
ancient individuals from West Eurasia, defined here as the region west of the
Ural Mountains (longitude, <60° E) and north of 35° N (SI Appendix, Fig. S1).
We excluded samples with less than 0.1× coverage. We removed duplicate and
closely related samples (e.g., first-degree relatives), by calculating pairwise
identity by state (IBS) between ancient individuals. For each individual, we
identified a corresponding individual with the highest IBS. Based on the dis-
tribution of highest IBS values, we identified the pairs with IBS Z score >7 as
duplicates and retained the individual with fewest missing sites. We also
manually removed related samples that were explicitly annotated, selecting
the highest coverage representative. Finally, we incorporated additional an-
cient samples from the Iberian Peninsula from two recent papers (36, 45),
applying the same criteria for coverage and relatedness. Ultimately, we com-
piled a list of 1,158 ancient pseudohaploid individuals in our analyses covering
a time span of the last 40,000 y. Only 11 individuals were dated to >15,000 y
BP, and 1,147 individuals lived during the last 15,000 y.
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Shotgun Ancient DNA Dataset. We organized a dataset of samples that were
shotgun sequenced without enrichment using the capture array (23, 25–32,
35, 39, 42, 45, 47–51, 54, 56, 58, 60–67). To generate the first 10 PCs from
sites on the 1240K capture array, we combined shotgun samples with
available genotype data from v37.2 of the Reich laboratory dataset and 15
individuals that we ourselves pulled down from BAM files. We manually
removed duplicate samples in the shotgun dataset, preserving the sample
with higher coverage. We checked for first-degree relatives in the dataset by
considering familial annotations but found none. The final shotgun dataset
included 249 individuals.
Skin Pigmentation SNP Curation. We obtained summary statistics for UK
Biobank GWAS for skin color (data field 1717) from the publicly available
release by the Neale Lab (version 3, Manifest Release 20180731) (14). The
GWAS measured self-reported skin color as a categorical variable (very fair,
fair, light olive, dark olive, brown, black). To identify genome-wide signifi-
cant and independent SNPs, we performed clumping using PLINK v1.90b6.6
(89) with 1000 Genomes GBR as an LD reference panel (--clump-p1 5 × 10−8
--clump-r2 0.05 --clump-kb 250), and followed up with clumping based on
physical distance to exclude SNPs within 100 kb of each other. We made two
separate lists of UK Biobank SNPs for the shotgun and capture-shotgun
datasets because the capture-shotgun dataset was restricted to the 1240K
array sites. For the capture-shotgun dataset, we intersected all UK Biobank
SNPs with 1240K array SNPs before identifying 170 independent and
genome-wide significant SNPs (Dataset S1A), which were used in Figs. 1 B
and C, 2 B and C, 3, and 4. For the shotgun dataset, we identified 242 in-
dependent and genome-wide significant SNPs (Dataset S1B), which were
used in Figs. 1A and 2A. For SI Appendix, Fig. S4, we identified 93 SNPs from
the Neale Lab GWAS (Dataset S1F) by picking the top SNPs that are genome-
wide significant from nearly independent LD blocks in the human genome
specified beforehand (62). For SI Appendix, Fig. S3, we identified 176 SNPs
(Dataset S1G) using the same clumping parameters with UK Biobank GWAS
summary statistics calculated with fastGWA (61).
We also manually curated a list of skin pigmentation-associated SNPs from
the literature (Dataset S1C). We identified 12 suitable studies (12, 13, 15, 80,
90–97), 9 of which were GWAS conducted in diverse populations (Dataset
S1D). We did not include SNPs from all of the considered papers for our
analysis because some studies reported associations that were not genome-
wide significant (P < 5 × 10−8). However, we included subsignificant SNPs
6 of 8 | PNAS
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from a GWAS in Europeans (95) with evidence of replication (P < 5 × 10−8) in
the UK Biobank GWAS. For a GWAS in African populations (12), we selected
a SNP for each LD block, which was defined by the study authors, with the
greatest support based on multiple lines of evidence (e.g., P value and
functional annotations). For a GWAS in South Asians (94), we picked SNPs
that were 200 kb away from each other and based the selection of SNPs that
were in physical proximity by considering P value. The other studies pre-
sented a set of independent SNPs that we considered for inclusion. Lastly, we
manually clumped the SNPs curated across studies, selecting SNPs 200 kb
apart with more statistical and/or functional evidence than nearby SNPs.
Specific details about the choices made on which SNPs to exclude can be
found in the “Notes” column for highlighted SNPs in Dataset S1C. We ended
with a final list of 18 SNPs for the manually curated set (Dataset S1E), 10 of
which are present on the 1240K capture array. We also made pseudohaploid
calls for each of the 18 SNPs in the shotgun dataset, picking a random allele
from the reads at each site as for the overall dataset.
ADMIXTURE Analysis on Capture-Shotgun Data. We performed unsupervised
ADMIXTURE (98) on the dataset of ancient individuals with K = 3, which we
found to produce the lowest cross-validation error for 2 < K < 9. The three
identified clusters could easily be identified as corresponding to hunter-
gatherer, Early Farmer, and Steppe (also referred to as Yamnaya) ancestry
(SI Appendix, Fig. S8).
Time Series Analysis of Genetic Scores. We calculated genetic scores for
each individual from present-day 1000 Genomes populations (67) and an-
cient individuals. We computed the scores in two ways. First, we weighted
by the GWAS-estimated effect sizes. Because of variable coverage
across ancient samples, not all SNPs were present in a given sample. To
account for missing information in the creation of weighted scores, we
devised a weighted proportion in which we divided the realized score over
the maximum possible score given the SNPs present in the sample:
Scoreweighted = ∑m m
i=1 di βi =∑i=1 βi , where m is the number of skin pigmentation
SNPs genotyped in the individual, di is the presence of the dark allele at the
ith SNP, and βi is the GWAS-estimated effect size (Fig. 1 A–C). For the
manually curated list of SNPs where we did not have comparable effect size
estimates, we computed an unweighted score, effectively assuming that all
variants had the same effect size. This score is the proportion of dark alleles
an individual carries out of the SNPs used in the construction of the score:
Scoreunweighted = (1=m)∑m i=1 di , where m is the number of SNPs genotyped in
the individual and di is the presence of the dark allele at the ith SNP
(Fig. 1 D–F).
We used logistic regression to examine the association between weighted
and unweighted genetic score and time for all ancient samples. We fitted
separate models for ancient samples in the shotgun and capture-shotgun
datasets. We included ancestry as a covariate in the model by including the
first 10 PCs. PCA was performed using smartpca v16000 (63) to generate PCs
from present day populations and we projected ancient individuals onto
these axes of variation. For the shotgun dataset and the capture-shotgun
dataset that included all samples (40,000 y BP), we used 1000 Genomes sam-
ples. For the capture-shotgun dataset that restricted to samples later than
15,000 BP, we used West Eurasians from the Human Origins dataset (16).
We model the score of each individual as the proportion of successes in a
binomial sample of size mi. That is, mi Si ∼ Binomial(mi , pi ), where, for indi-
vidual i, mi is the number of SNPs genotyped, Si is the (weighted or un-
weighted) score, the probability of success pi is given by the following:
pi
log( ) = α + βdate datei + βlat lati + βlon loni + βPC1 PC1i + . . . + βPC10 PC10i ,
1 − pi
and datei, lati, and loni are the dates, latitude, and longitude of each indi-
vidual. For the weighted score, miSi is noninteger, but the likelihood can be
computed in the same way.
For the stratified analysis, we divided the capture-shotgun dataset into
mutually exclusive ancestry groups. We categorized individuals for this
analysis as hunter-gatherer if they carried over 60% of the ADMIXTURE-
estimated hunter-gatherer component. For placement into the Early
Farmer group, we required that an individual carry over 60% of that com-
ponent. Individuals we categorized as Steppe had over 30% of the AD-
MIXTURE component and were dated to less than 5,000 y BP. Individuals
that are more recent than 5,000 y BP and have at least 30% of the Steppe
ADMIXTURE component were classified as Steppe even if they had more
than 60% of the Early Farmer component. Based on these cutoffs, there
were 102 hunter-gatherer, 499 Early Farmer, and 478 Steppe ancestry
Ju and Mathieson
The evolution of skin pigmentation-associated variation in West Eurasia
 assigned individuals. We used the same logistic regression model as above
for the stratified analysis to control for ancestry.
Because the fitted model parameters are overdispersed (SI Appendix, Fig.
S2), we computed P values from a genome-wide empirical null distribution
for βdate. We made this null distribution by running the regression model
described above on scores from sets of random, frequency-matched (±1%)
SNPs across the genome, maintaining the same effect sizes for the weighted
score. We matched the derived allele frequency based on the EUR super-
population frequencies from 1000 Genomes. We reported P values based on
10,000 random samples.
Time Series Analysis of Individual SNP Allele Frequencies. We performed lo-
gistic regression for each individual SNP using date of the sample and ancestry
as covariates. That is, the probability that the haplotype sampled from in-
dividual carries the derived allele is pi, where
pi
log( ) = α + βdate datei + βPC1 PC1i + . . . + βPC10 PC10i .
1 − pi
We compared the full model above to a nested model with no principal
components by performing a likelihood ratio test [in R we use anova(nested
model, full model, test = ‘Chisq’)] to obtain a P value for the ancestry term
which encompasses βPC1. . .βPC10. To obtain a P value for βdate, we compare a
nested model without βdate to the full model.
Qx Polygenic Selection Test. We used the Qx test for directional, polygenic
selection on skin pigmentation (10). For this test, we used 170 skin pig-
mentation SNPs obtained from the UK Biobank in all 1000 Genomes pop-
ulations. We restricted sites to those on the 1240K and constructed a
covariance matrix from a total of one million SNPs. To calculate empirical P
values, we sampled a total of 500,000 null genetic values matching skin
pigmentation SNPs based on a ±2% frequency on the minor allele. However,
we report one million runs for the top seven SNPs and two million runs for
the top five and six to better distinguish the deviation of the tested SNP sets
from the expectation under drift.
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Variance in Skin Pigmentation Explained by Individual SNPs. We estimated the
variance explained in the phenotype by a SNP within the UK population using
the following:
VEwithin = 2β2 f (1 − f ),
where β is the UK Biobank GWAS-estimated effect size and f is the allele
frequency in the GBR population from 1000 Genomes. We calculated the
proportion of variance explained as follows:
VEwithin
PVEwithin = ,
VEwithin + 2Nσ 2 f (1 − f )
where N is the GWAS sample size (356,530) and σ is the SE of the estimated β
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ST =
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,
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EVOLUTION
T X,YRI + T X,CHB − T YRI,CHB
PBSX = .
2
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Code Availability. Scripts used to generate the main results and figures of this
paper are available at GitHub, https://github.com/mathilab/SkinPigmentationCode.
ACKNOWLEDGMENTS. We thank Sarah Tishkoff, Alexander Platt, Bárbara
Bitarello, Samantha Cox, Arslan Zaidi, and Andrew Chen for helpful com-
ments on earlier versions of this manuscript. This research was supported by
a Research Fellowship from the Alfred P. Sloan Foundation (FG-2018-10647),
a New Investigator Research Grant from the Charles E. Kaufman Foundation
(KA2018-98559), National Institute of General Medical Sciences Award
R35GM133708, and National Research Service Award T32GM008216. The
content is solely the responsibility of the authors and does not necessarily
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Ju and Mathieson
The evolution of skin pigmentation-associated variation in West Eurasia

rspb.royalsocietypublishing.org
Research
Cite this article: Council SE, Savage AM,
Urban JM, Ehlers ME, Skene JHP, Platt ML,
Dunn RR, Horvath JE. 2016 Diversity and
evolution of the primate skin microbiome.
Proc. R. Soc. B 283: 20152586.
http://dx.doi.org/10.1098/rspb.2015.2586
Received: 27 October 2015
Accepted: 1 December 2015
Subject Areas:
evolution, microbiology
Keywords:
microbiota, microbe, microbiome,
primate, skin, axilla
Author for correspondence:
Julie E. Horvath
e-mail: julie.horvath@naturalsciences.org
Electronic supplementary material is available
at http://dx.doi.org/10.1098/rspb.2015.2586 or
via http://rspb.royalsocietypublishing.org.
Diversity and evolution of the primate
skin microbiome
Sarah E. Council1,3, Amy M. Savage4, Julie M. Urban3, Megan E. Ehlers3,
J. H. Pate Skene5, Michael L. Platt5,6,7,8, Robert R. Dunn9,10
and Julie E. Horvath2,3,11
1
Center for Science, Math and Technology Education, and 2Department of Biological and Biomedical Sciences,
North Carolina Central University, Durham, NC 27707, USA
3
North Carolina Museum of Natural Sciences, Raleigh, NC 27601, USA
4
Department of Biology, Center for Computational & Integrative Biology, Rutgers University, Camden,
NJ 08103, USA
5
Department of Neurobiology, Duke University, Research Drive, Durham, NC 27710, USA
6
Department of Neuroscience, Perelman School of Medicine, 7Department of Psychology, School of Arts and
Sciences, and 8Department of Marketing, the Wharton School, University of Pennsylvania, Philadelphia,
PA 19104, USA
9
Department of Applied Ecology and Keck Center for Behavioral Biology, North Carolina State University,
Raleigh, NC 27607, USA
10
Center for Macroecology, Evolution and Climate, Natural History Museum of Denmark, University of
Copenhagen, Copenhagen Ø 2100, Denmark
11
Department of Evolutionary Anthropology, Duke University, Durham, NC 27708, USA
Skin microbes play a role in human body odour, health and disease. Compared
with gut microbes, we know little about the changes in the composition of skin
microbes in response to evolutionary changes in hosts, or more recent behav-
ioural and cultural changes in humans. No studies have used sequence-based
approaches to consider the skin microbe communities of gorillas and chimpan-
zees, for example. Comparison of the microbial associates of non-human
primates with those of humans offers unique insights into both the ancient
and modern features of our skin-associated microbes. Here we describe the
microbes found on the skin of humans, chimpanzees, gorillas, rhesus maca-
ques and baboons. We focus on the bacterial and archaeal residents in the
axilla using high-throughput sequencing of the 16S rRNA gene. We find
that human skin microbial communities are unique relative to those of other
primates, in terms of both their diversity and their composition. These differ-
ences appear to reflect both ancient shifts during millions of years of primate
evolution and more recent changes due to modern hygiene.
1. Introduction
The skin is the largest mammalian organ [1,2], and the primary physical barrier
between mammals and the outside world. The outermost layer of the skin com-
prises host cells along with billions of microbial symbionts. Skin microbes
inhibit the colonization of opportunistic or pathogenic microbiota [3,4], regulate
immune activation [5–7], and produce compounds that function as both phero-
mones [8,9] and allomones [10]. Skin microbes also influence the attractiveness
of hosts to blood-feeding insects, including mosquitoes, known to transmit
malaria, dengue and chikungunya [11 –14]. The significance of skin microbes
to human health and disease suggests that the features of the body that influ-
ence its composition might coevolve with host traits. On human skin, the
abundance and composition of microbes varies relatively predictably among
habitats on the body, as a function of many factors including the distribution
of glands and moisture [15 –17]. For example, the apocrine glands in the axillary
organ in the armpit produce secretions that provide food for the microbes living
therein [18 –20]. Microbial constituents of the skin microbe communities play an
important role in odour creation due to their production of volatile organic
compounds [18,19]. Body odour plays a central role in primate society in the
& 2016 The Author(s) Published by the Royal Society. All rights reserved.
context of mating, child rearing, predatory protection and ter- 30 CGBR HBR HR HC HC 2
ritorial marking [21,22]. While we are beginning to learn which

rspb.royalsocietypublishing.org
host and environmental factors influence skin microbes, we

no. OTUs (transformed)

know little about how the microbial composition of the skin
varies among closely related primates. 20
The composition of microbes on human skin might be
expected to differ significantly from that of our closest relatives,
the non-human primates, for at least three reasons. First, even 10
closely related primates differ in the distribution and abun-
dance (and likely chemical composition) of different skin
glands. Skin glands provide both food sources and habitat
for microbes [23–27]. For example, our closest evolutionary 0

Proc. R. Soc. B 283: 20152586

an

ee

la

on

e
relatives, chimpanzees and bonobos, share the majority of

u
ril
nz
m

aq
bo
go
hu

pa

ac
ba
our nuclear genome [28], yet differ in their abundance and

im

sm
ch
location of eccrine and apocrine glands [26,29] (see the elec-

tronic supplementary material, text), the form of sebaceous
gland exudate [25] and the expression of immune-related
genes compared with humans [30]. The non-human apes, in
turn, differ from the Old World monkeys (e.g. rhesus macaques
and baboons, approx. 30 Myr divergent from the apes) [31] in
other attributes of similar features. For example, while humans,
chimpanzees and gorillas have apocrine glands clustered in the
axillae (armpits), in Old World monkeys apocrine glands are
not clustered and can be found (typically at lower densities)
covering the entire body and in equal number to eccrine
glands [26,29] (electronic supplementary material, text).
Second, over the last 100 years, human hygienic behaviour has
dramatically changed such that the skin of most humans is
now exposed daily to soaps, detergents and underarm products,
some of which affect skin microbes [16,32,33]. Third, microbes
might differ among primate hosts as a function of their evol-
utionary dissimilarity [34,35], whether as a result of drift or
selection on host traits that influence skin microbes. Collectively,
then, we might predict differences in the skin microbiotas among
primate hosts, including humans, that reflect both the recent
shifts in human hygiene and more ancient divergences in the
biology of the skin in addition to host evolutionary history.
Specifically, we might predict large differences between the
monkey and the ape hosts, and, within the apes, more subtle
differences between human and non-human apes.
The skin habitats with high microbial abundance and
functional consequences (e.g. on mating or social behaviour)
are perhaps the most likely to be strongly influenced by host
evolution (akin to the situation in the gut [34]). The axillae
have one of the highest bacterial biomasses on the skin sur-
face [36,37] and are directly sustained by food resources
primarily provided by apocrine glands [18]. If the influence
of evolution is to be apparent on any part of the skin micro-
biome, it should be in the axillae, especially given the known
differences in apocrine gland distribution and density among
primates (electronic supplementary material, text).
Here we assess the microbial (bacterial and archaeal) com-
munities in the axillae of five primate species. Samples were
collected from humans, zoo populations of chimpanzees, gor-
illas and baboons, and semi-free-ranging rhesus macaques. We
used high-throughput pyrosequencing of the 16S rRNA gene
to identify microbes present in primate axillae. We hypo-
thesized that the composition and diversity of axillary
microbes would be different for each host species and pre-
dicted that their composition would track the phylogenetic
relatedness of primate hosts. Exclusive of studies focused on
humans, our study is the first to consider the microbes on pri-
mate skin with high-throughput sequencing methods. In doing
u
es
rh
Figure 1. Number of OTUs per primate species after rarefying to 1000
sequence reads per individual and square-root transforming. In order to fit
the assumptions of an ANOVA, square-root transformations were conducted
because of unequal variances. Levene’s Test, one-way ANOVA and Tukey’s
multiple comparison test were completed on the mean of each host group
(F4,62 ¼ 51.3, p , 0.05). Letters above columns correspond to the first
letter of the primate host that is significantly different ( p , 0.05).
so, we reveal a clear signature of host evolution and biology
on the microbiome of the axillae. These results emphasize the
role of evolutionary relationships in defining the human skin
ecosystem, and have implications for the role of skin microbes
in health and disease.
2. Results and discussion
(a) Axillary microbial richness
We obtained ribosomal RNA gene sequences from 63 samples
of primate axillae using high-throughput pyrosequencing
(Roche 454), and processed them using the Quantitative
Insights into Microbial Ecology (QIIME) pipeline [38]. To evalu-
ate microbial diversity, QIIME categorizes and groups similar
sequences that share a threshold level of sequence identity
into a defined operational taxonomic unit (OTU); it then ident-
ifies a representative sequence from each OTU group to assign
taxonomic lineage. Using a 97% nucleotide-sequence identity
threshold to define OTUs, we narrowed our samples to a
single axilla sample per individual and rarefied to 1000
sequences per individual (see Methods; electronic supplemen-
tary material, figure S1). We identified 5309 unique OTUs
from our primate axillae samples; 30 OTUs were unclassified
(probably sequencing artefacts), 15 were archaeal in origin
and 5264 were bacterial OTUs.
Using the rarefied dataset, we found that humans had the
lowest average richness of microbial OTUs per individual
(one-way ANOVA and subsequent pairwise testing using
Tukey’s multiple comparison test: p , 0.05; figure 1). Chim-
panzees and gorillas hosted a similar richness of skin
microbes and each had a significantly lower richness of
skin microbial OTUs than did rhesus macaques (one-way
ANOVA and Tukey’s tests: p , 0.05; figure 1). In addition,
chimpanzees had significantly higher OTU richness than did
baboons (one-way ANOVA and Tukey’s tests: p , 0.05;
figure 1). Furthermore, the axillae of rhesus macaques and
baboons had a significantly higher OTU richness than did
those of humans and chimpanzees (one-way ANOVA and
Tukey’s tests: p , 0.05). In general, the higher OTU richness of
skin microbes in monkeys relative to apes is in line with expec-
tations if the increased number of axillary apocrine glands in
apes [29] tends to favour a subset of bacterial taxa at the expense
of diversity (electronic supplementary material, text).
The four most common human skin bacterial phyla—
Firmicutes, Actinobacteria, Proteobacteria and Bacteroidetes
[8]—comprised over 97% of the sequence reads in the human
axillae samples (electronic supplementary material, figure
S2), similar to that observed in studies focused on human
skin microbes [15]. These same phyla made up 85% of the
sequence reads in chimpanzees and gorillas, 82% in baboons
and 88% in rhesus macaques (electronic supplementary
material, figure S2).
(b) Composition of axillary microbes among hosts
To understand the microbial composition among host
primates, we first assessed the underlying compositional data
matrix (sequence count of each microbial genus by individual
host) to confirm that there was structure in the dataset using the
KR-means with a Simprof significance test in PRIMER-E v. 7.0.8.
We found that there was significant structure in microbial com-
position among hosts (R ¼ 0.91, p ¼ 0.01). We then applied a
hierarchical approach to assess whether the composition of
skin microbes differed predictably among primate groups
(monkeys and apes), within each group (monkeys: baboons,
rhesus macaques; apes: humans, chimpanzees, gorillas). We
then examined the individual variation in skin microbial com-
position within host species. Finally, we compared the skin
microbes of human zoo workers with other humans and
to apes living in zoos to explore proximity transfer of microbes.
We visualized relative composition per host using non-
metric multi-dimensional scaling (NMDS) and employed
PerMANOVA (a permutational ANOVA/MANOVA) and
PermDISP ( permuted dispersion, which tests for homogeneity
of dispersions) to statistically evaluate differences among hosts
(see Methods).
After demonstrating overall structure by microbial compo-
sition of host axillae, we tested primate groups and found
that the composition of axillary microbes differed significantly
between monkeys and apes (PerMANOVA, p , 0.01; figure 2).
These differences were primarily driven by Corynebacterium
and a genus of Staphylococcaceae (not further differentiated
using the 16S rRNA segment we sequenced, so hereafter
referred to as Staphylococcaceae), both of which were much
more abundant in apes than in monkeys; both Corynebacteria
and Staphylococcaceae/Staphylococcus have also been found
in high abundance in studies of human skin microbes
[15,20,36,39–42]. Together, Corynebacteria and Staphylococca-
ceae contributed to more than 37% of differences among apes
and monkeys (SIMPER analysis; table 1). Interestingly, a
small number of individual gorillas and chimpanzees had
skin microbes that were compositionally more similar to mon-
keys than was the case for the average gorilla or chimpanzee
(figure 2; see circles near triangles). These individual hosts
had lower abundances of Corynebacterium than other apes
(unpaired t-test: p , 0.01), but no significant difference in
Staphylococcaceae (unpaired t-test: p . 0.05). Despite such
differences in microbe composition among individuals
within host species, the skin microbes on different host species
generally tracked the evolutionary history of those hosts, just as
microbe composition by host species 3
1.50 2D stress = 0.14 PerMANOVA: p = 0.0001
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0.75
axis 2
0
–0.75
Proc. R. Soc. B 283: 20152586
–1.50
–1.50 –0.75 0 0.75 1.50
axis 1
rhesus macaques
baboons
gorillas
chimpanzees
humans
Figure 2. NMDS plot of the composition of skin microbial communities across all
five primate species. Each point represents an individual host; triangles represent
monkeys (baboons and rhesus macaques), while circles represent apes (gorillas,
chimpanzees and humans). Species are coded with different colours. Larger sym-
bols represent the centroid for each species + 1 se. The composition of skin
microbiota on monkeys was significantly different from that of apes (PerMANOVA:
p , 0.01). Within monkeys, baboons and rhesus macaques had significantly
different skin microbiota (PerMANOVA: p , 0.01). Within apes, gorillas and
chimpanzees had significantly different skin microbiota than humans (PerMA-
NOVA: p , 0.01 for both comparisons); however, the composition of gorilla
and chimpanzee skin microbiota did not differ significantly from each other (Per-
MANOVA: p ¼ 0.12). (Online version in colour.)
has been shown for gut and saliva microbiomes [35,43]
(electronic supplementary material, figure S3a,b).
When we investigated differences among individuals within
primate groups, we found that within monkeys, there were
significant differences between the skin microbiota in the axillae
of baboons and in those of rhesus macaques (PerMANOVA, p ¼
0.01; figure 2). Prevotella contributed to 10% of the total differ-
ences between the two monkey hosts, while Staphylococcaceae
was responsible for 8% of the total differences between baboons
and rhesus macaques (electronic supplementary material,
table S1). Prevotella, an anaerobic group of microbes found in
the mouth, vagina and gut of humans, represented the most
common taxon (22% of all reads; figure 3, green block) in the axil-
lae of rhesus macaques. The abundance of Prevotella was greater
in the axillae of rhesus macaques compared with baboons
(one-way ANOVA: p , 0.05; electronic supplementary material,
figure S4). The composition of microbes in baboon axillae
was diverse. The most common taxon was Staphylococcaceae,
which accounted for 12% of reads (figure 3, red block). In con-
trast to differences in composition within hosts, there were no
significant differences in beta diversity (a measure of variability
among groups) between baboons (zoo animals) and rhesus
macaques (semi-free-ranging; PermDISP: p ¼ 0.89; figure 4).
This is somewhat surprising given that differences in microbial
composition have been previously noted between captive and
wild host species [43,44].
Within apes (gorillas, chimpanzees and humans), the axil-
lary microbial communities of humans were significantly
Table 1. Comparison of the average abundances (number of sequence reads) and percentage contributions of the 10 microbial taxa that contributed the most 4
to differences between apes and monkeys (SIMPER analysis, PRIMER-E v. 7.0.8). Undetermined microbial taxa (each of which is a single OTU) are those that did

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not match sequences for known, named taxa at genus level. In some cases, with longer sequence reads, these taxa (e.g. Staphylococcaceae) might correspond
with known phyla, classes, genera and species, respectively.

average abundance
% contribution to % cumulative
comparison family genus apes monkeys differences contribution

apes versus Staphylococcaceae undetermined 0.37 0.08 20.79 20.79

monkeys Corynebacteriaceae Corynebacterium 0.29 0.04 16.56 37.35

Proc. R. Soc. B 283: 20152586

Prevotellaceae Prevotella 0.02 0.09 4.77 42.11
Clostridiaceae Anaerococcus 0.06 0.01 3.86 45.97
undetermined phylum 0.01 0.07 3.61 49.58
Pasteurellaceae undetermined 0.00 0.03 1.89 51.47
Streptococcaceae Streptococcus 0.00 0.03 1.83 53.30
Ruminococcaceae Ruminococcus 0.01 0.02 1.58 54.88
undetermined Actinomycetales 0.00 0.03 1.41 56.29
undetermined Streptophyta 0.01 0.02 1.33 57.62

100 50
A
AB
(among-host dissimilarity)

80 B
percentage of total reads

40
beta diversity

60
40
20
0
human chimpanzee gorilla
host primate species
Corynebacterium
Staphylococcaceae
Prevotella
Anaerococcus
other microbial species
Figure 3. Percentage of total reads of the Staphylococcaceae family, and
genera of Corynebacterium, Anaerococcus and Prevotella in each of the five
host primate species. Columns represent average sequence reads per individ-
ual within host group.
different from those of chimpanzees and gorillas (PerMA-
NOVA: p , 0.01 for both). However, there were no significant
differences in the composition of skin microbiota between
chimpanzees and gorillas (PerMANOVA: p ¼ 0.12; figure 2;
electronic supplementary material, table S1). Greater abun-
dances of Corynebacterium and Staphylococcaceae in humans
than in other apes underlie most of this divergence in axillary
microbiota between humans and other apes. These two
genera contributed 52% of the total compositional differences
between humans and chimpanzees, and 41% of the total com-
positional differences between humans and gorillas
(electronic supplementary material, table S1). The abundance
of Staphylococcaceae was significantly higher in humans com-
pared with all other primate hosts (one-way ANOVA: p , 0.05;
30
20
10
baboon rhesus macaque
0
human chimpanzee gorilla baboon rhesus macaque
apes monkeys
Figure 4. Beta diversity bar charts showing dissimilarity in microbial content
(based on number of sequences and taxonomic classification) between
primate species within apes and monkeys. Within apes, humans and gorillas
were significantly different (PermDISP: p , 0.05). There was no significant
difference between humans and chimpanzees or chimpanzees and gorillas.
Within monkeys, there were no significant differences between baboons
and rhesus macaques.
electronic supplementary material, figure S4). Corynebacterium
abundance was significantly higher in humans and chim-
panzees when compared with baboons (one-way ANOVA:
p , 0.05; electronic supplementary material, figure S4).
Humans had the highest level of compositional variability
from one individual to the next, in line with the expectation
if human underarm products have a strong influence on the axil-
lary microbiome. Recent studies investigating the effects
of underarm product use found shifts from Corynebacterium to
Staphylococcaceae in people who use underarm products such
as antiperspirant and deodorant [32,33]. Additionally, people
who routinely wore no product hosted higher abundances
of Corynebacterium than of Staphylococcaceae [32]. Here, our
results recontextualize those findings and suggest that in the
absence of deodorant and antiperspirant, humans have axillary
microbes more similar to apes (more Corynebacterium, less
Staphylococcaceae), but that by altering the relative abundance of
these taxa, underarm product use makes us less similar to
the other apes (electronic supplementary material, table S1).
Within host species, the composition of microbes tended to
vary more from one individual to the next within ape species
versus within monkey species (PermDISP, p , 0.01; electronic
supplementary material, figure S5).
Given that the skin is constantly in contact with the sur-
rounding environment, and in the light of recent evidence of
transfer of microbes during contact sports [45], we investigated
whether people who came into contact with non-human pri-
mates acquired microbes from the animals they worked with.
Individuals who worked with non-human primates did not
differ from other humans in terms of their axillary microbiome,
nor did they differ from non-human apes (electronic sup-
plementary material, text and figure S6a,b). Thus, if transfer
occurs between humans and non-human animals it does not
eclipse the differences intrinsic to host species [43], but it
may be associated with more subtle shifts (hence our inability
to distinguish those with close contact with other primates
compared with none).
(c) A core primate axillary microbiome
Despite the differences among individual hosts and among
host species, we identified a core primate axillary microbiome
comprising taxa shared by at least 95% of individuals [46].
OTUs of the genus Corynebacterium were found in all primate
individuals sampled. Corynebacterium, Prevotella, Anaerococcus
and a genus of Staphylococcaceae were also among the most
abundant taxa based on read number (figure 3; electronic
supplementary material, figure S7). These taxa are consist-
ently frequent (found on many individuals) and abundant
( present as many reads). Meanwhile, the diversity of OTUs
outside of the top four genera increased with evolutionary
distance from humans (figure 3; electronic supplementary
material, figure S7), an effect likely to be due to a complex
mix of the influence of human hygiene, the evolutionary his-
tory of the axillary organ and other host differences that have
accrued over evolutionary time. Among the most common
taxa in the primates most evolutionarily distant from
humans (in our sampling), the monkeys, were OTUs of
Prevotella and Actinomycetales. In addition, the monkeys
have a large number of microbial genera (more than 30
genera) in their core axillary microbiome that are generally
associated with soil, gut and oral microbiomes such as Acine-
tobacter, Ruminococcaceae and Porphyromonas, respectively. It
is unclear whether these taxa live on the skin persistently, or
represent frequent contamination of the skin with soil and
faeces (or both).
3. Conclusion
A large body of recent research has suggested that human
exposure to microbes has changed dramatically over the last
100 or 200 years (and prior to that, with the origin of agriculture
more than 10 000 years ago), with consequent shifts in gut [35]
and oral microbes [43], in extreme cases leading researchers
to the ‘hygiene hypothesis’ [47,48]. Here, we find that the
shift is variable among individuals, and rather than being
associated with sweeping changes in microbial fauna, it is
associated with two key changes. First, humans seem to be
less covered with faecal and soil microbes than are other
primates, particularly monkeys. Second, humans have a less 5
diverse skin microbial community, more dominated by Sta-
rspb.royalsocietypublishing.org
phylococcaceae than is the case for any other primate
sampled. The latter is interesting in as much as Staphylococcus
has long been viewed as the ‘normal’ skin symbiont. It is also
one that has been noted to attract mosquito species, including
at least one vector of malaria [11]. Our work suggests the
modern composition of our axillary microbiome, including
the abundance of Staphylococcaceae, is new at least relative
to our divergence with other primates. Whether it is new rela-
tive to changes in the last few hundred years remains to be
seen. Nonetheless, our findings advance our understanding
Proc. R. Soc. B 283: 20152586
of the evolutionary context of the diversity of human skin
microbiota, and suggest that phylogenetic relatedness among
hosts may strongly influence the composition of skin micro-
biomes across all primates.
4. Methods
(a) Sample collection
Samples from both the left and right human axillae of participants
were collected between August 2012 and May 2013. Human volun-
teers from the North Carolina Museum of Natural Sciences
(NCMNS) and North Carolina State University (NCSU) were
asked to refrain from using deodorant or antiperspirant for 2
days prior to sampling, but were allowed to shower normally. Par-
ticipants were told to swab each axilla with a sterile dual-tipped
rayon swab (BD BBL Cat no B4320135) for at least 45 s while rotat-
ing the swab so that all sides of each swab came in contact with the
axillary skin. In this study, we engaged 20 human participants
from a concurrent study, in addition to 17 collected specifically
for this study, for a total of 37 human participants. We collected
samples from non-human primates during routine physicals of
baboons, chimpanzees and gorillas at the North Carolina Zoo,
and rhesus macaques in Cayo Santiago in Puerto Rico. All zoo pri-
mates were captive born except for one chimpanzee born in the
wild. None of the gorillas were taking antibiotics at the time of
swabbing, but all of the gorillas had been treated within the past
six months with a dewormer (mebendazole or pyrantel pamoate).
None of the other non-human primates were being given any
medication. All zoo primates had outdoor and soil access, and
ate vegetarian diets. The rhesus macaques lived entirely outdoors
and were rationed with monkey chow. In total, we sampled
11 baboons, seven chimpanzees, five gorillas and two rhesus
macaques. Swabs were stored at 48C until processed. Human
samples were processed within weeks of initial sampling; primate
samples were processed within months after initial sampling.
(b) DNA extraction and pyrosequencing
Each armpit was swabbed with a dual-tipped rayon swab. One
of each of these tips was used for qualitative plating for outreach
events at NCMNS; the second was used for DNA extraction for
pyrosequencing (454 Roche Genome Sequencer FLX system).
DNA was extracted with the PowerSoil DNA Isolation Kit (MO
BIO Laboratories, Carlsbad, CA, USA) following manufacturer’s
protocol with a modification of a two-step elution process into 50
total microlitres of elution buffer C6. DNA was stored at – 208 C
until amplified and sequenced. Isolated DNA was PCR amplified
with 16S rRNA primers (515F and 806R(49)) with 454 adapters
and index sequences using Premix Ex Taq (Takara Bio, Siga,
Japan) [38]. PCR amplification was done as follows: initial dena-
turation at 948C for 2 min, five cycles of 948C for 20 s, 538C 30 s,
728C for 1 min, then 30 cycles of 948C for 20 s, 558C for 30 s then
728C for 1 min followed by a final 728C extension for 7 min and a
108C hold. Each host individual had a unique index and PCRs
were run with 3 – 6 ml of isolated microbial DNA in triplicate in
25 ml reactions with 1.25 mM each primer with a 1X PCR
master mix. PCRs were visualized using gel electrophoresis
and then triplicate reactions from each individual were pooled
and purified using UltraClean-htp 96-well PCR Clean-up kit
(MO BIO) per manufacturer’s protocol. An equal mass of
purified pooled product, as measured by Qubit dsDNA HS
Assay Kit (Invitrogen, Eugene, OR, USA), was added to a
single tube and then ethanol precipitated to concentrate the mix-
ture. The ethanol-precipitated 16S rRNA pooled mixture was
measured by Qubit and was sent for Roche 454 next-generation
pyrosequencing (Selah Genomics, Greenville, SC, USA).
(c) Sequence analysis
In addition to the 17 newly collected samples taken from non-
human primates and humans for this study, we included data
from 20 additional human participants collected for a different
study [32]. Combining these two datasets yielded 798 818 sequence
reads with 698 799 passing quality and length filters. Amplicons of
the V4–V5 region [49] of the 16S rRNA gene were processed and
analysed using the QIIME pipeline (MacQIIME 1.7.0-20130523;
electronic supplementary material, text) [38]. DNA sequences
were processed, filtered and assigned to individuals according to
a 13 bp barcode, derived from the standard 12 bp barcode [49]
with one bp added in silico to all samples in the mapping file
and the original fna files in order to combine the Urban et al.
samples that used the same 12 bp barcodes. Denoising of samples
was not done due to flow pattern B processing of 454 sequence
data, which enabled longer reads but removed the denoizing capa-
bility. OTUs were clustered by UCLUST v. 1. 2.22q [40] using a 97%
nucleotide similarity threshold such that reads 3% different (or
less) from each other were assigned to the same taxon. Taxonomic
identities were aligned using PYNAST v. 1.2 and assigned based
off GREENGENES v. 12-10) [50,51]. OTUs of the family Staphylococca-
ceae were common in our dataset (more than 50% sequences), but
could only be defined at the family-level classification. The OTUs
of the family Staphylococcaceae taxon fell within a single 97%
de novo OTU, thus we reference them in comparison to other
genus-level OTUs. All samples were rarefied to 1000 sequence
reads per sample prior to downstream analyses. Five samples
were removed from analysis due to limited read depth. Analysing
53 individuals with both left and right samples, there was no sig-
nificant difference (two-tailed unpaired t-test: p ¼ 0.81) in genus-
level OTU counts in left versus right axilla samples. Because we
found no significant difference between left and right axilla
samples, and some host individuals provided only one axillary
sample, we analysed a single axillary sample for each individual
in this study (electronic supplementary material, figure S1). For
individuals providing both axillary samples, one side was
chosen based on the highest genus-level OTU value by sample
per individual. Data from both axillae are deposited in NCBI as
Bioproject PRJNA281417 (http://www.ncbi.nlm.nih.gov/biopro-
ject/; electronic supplementary material, text).
(d) Operational taxonomic unit data processing
All further data analysis used only one axillae sample per partici-
pant. The homogeneity of variances of host OTUs was assessed
using Levene’s test (IBM SPSS Statistics for Windows, v. 23.0;
Armonk, NY) to determine whether the data fit the assumption
of an ANOVA. After square-root transformation, a one-way
ANOVA and Tukey’s multiple comparison test were used to com-
pare the mean of the square root transformed OTUs in each host
group using GraphPad PRISM v. 5.01 (La Jolla, CA, USA). Further
downstream analysis was calculated using QIIME output at the
phylum level (L2) or the genus level (L6). Sequence reads and
abundance datasets are available on FigShare under the manu-
script title (Figshare.com).
(e) Clustering via KR-clustering 6
Before conducting statistical analyses of the composition data, we
rspb.royalsocietypublishing.org
used KR-clustering in PRIMER v. 7.0.8 [52] to identify whether
structure existed with regard to the differences in microbe species
composition among individual hosts and host species at the
genus level. We constructed a dissimilarity matrix, in which
microbial abundances were compared between samples. With
this matrix, we conducted the KR-cluster analysis using 100
restarts and a Simprof significance test with two to five groups
and a ¼ 0.001. Individual hosts clustered more than would be
expected based on chance. On the basis of this result, we next
analysed the relationship between skin microbial composition
and attributes of hosts, which might explain this clustering.
Proc. R. Soc. B 283: 20152586
(f ) Analysis of skin microbe compositional diversity
We compared the composition of skin microbes among the axil-
lae of individuals of all five primate hosts; we tested whether the
differences among host species in the composition of microbial
communities were greater than the differences among host indi-
viduals within species using a PerMANOVA. All analyses of
composition, including Bray – Curtis dissimilarity, non-metric
multi-dimensional scaling plot, SIMPER analysis and PermDISP,
were conducted using PRIMER-E v. 7.0.8 with the PerMANOVA
ext. v. 1.0.3 [52]. We first constructed a resemblance matrix
based on the number of sequence reads of each microbe taxon
on each host individual using Bray– Curtis dissimilarities.
Bray – Curtis dissimilarity is a standardized metric of compo-
sitional dissimilarity among ecological samples, such as swabs
of axillae or quadrats in old fields. Values range between 0 and
1, with 0 indicating no difference among samples and 1 indicat-
ing that two samples do not share any microbial species [53].
Bray – Curtis dissimilarity has been recommended as a robust
measure of ecological distance for complex communities [54].
Next, we created a NMDS with a Type I Kruskal fit scheme
and 100 restarts; NMDS is a preferred approach when differences
among samples may be due to categorical variables (e.g. host
species) rather than continuous gradients (as in detrended
correspondence analysis) [55]. To test our a priori hypothesis
regarding the causes of differences in microbe composition
among hosts, we used PerMANOVA with the independent
factor of apes (humans, gorillas, chimpanzees) versus monkeys
(baboons, macaques; 9999 iterations, and Type III sums of
squares). When differences between apes and monkeys were
significant, we assessed pairwise differences among host species
within apes and within monkeys using PerMANOVA with
species as a factor, 9999 iterations and Type III sums of squares.
To determine the difference between zoo workers, other humans
and zoo animals (chimpanzees, gorillas and baboons), we per-
formed PerMANOVA with the independent factor HumanZoo
(human zoo workers þ humans working with wild primates,
other humans and non-human primates housed in zoos).
We performed SIMPER analysis to determine which OTUs
contributed the most to differences among host groups.
We assessed the variation in skin microbial composition
among host species using PermDISP. Specifically, we trans-
formed the data matrix into a presence/absence matrix, and
then constructed a resemblance matrix using Bray– Curtis dis-
similarities using these transformed values. Using this matrix,
we performed a PermDISP using host cluster as the independent
factor, 9999 iterations and centroids as the measure of central ten-
dency. Here, we were testing whether taxa differed in terms of
how variable composition was among individuals within the
host (e.g. among chimpanzees versus among gorillas). Again,
when differences between apes and monkeys were significant,
we assessed pairwise differences for each species pair within
apes and monkeys as described above.
 of the study and drafted the manuscript; A.M.S. carried out the stat-
(g) Core microbiome istical analyses and drafted the manuscript; M.E.E. collected field
7
Primate core microbe taxa were determined based on the QIIME L6
data; J.M.U. helped analyse data; J.H.P.S. and M.L.P. aided in data

genus-level output. Microbes found in more than 95% of all indi-
viduals sampled across all hosts were determined to be in the
primate core microbiome [46]. Microbes found in all non-human
primate species were termed non-human primate core microbes.
Any microbe found in all monkey individuals (baboons and
rhesus macaques) was considered to be a monkey core microbe.
Ethics. All human participants were provided a written informed con-
sent form approved by North Carolina State University Institutional
Review Board for the Protection of Human Subjects in Research (IRB)
(approval no. 1987) or North Carolina Central Institutional Review
rspb.royalsocietypublishing.org
collection and manuscript revisions; J.E.H. and R.R.D. conceived of
the study, designed the study, coordinated the study, participated
in data analysis and drafted the manuscript. All authors provided
valuable insights on the manuscript and gave final approval
for publication.
Competing interests. We have no competing interests.
Funding. This project was funded through PI R.R.D. (W911NF-
14-1-0556 from the Army Research Office) and is part of a
larger collaborative grant to PI M.L.P. (NIMH R01-1MH096875-
01A1).
Acknowledgements. We thank Dr Lauren Brent, Dr Richard Bergl, Chris

Proc. R. Soc. B 283: 20152586

Board (approval no. 1201121).
Data accessibility. DNA sequences: NCBI as Bioproject PRJNA281417.
Also on FigShare under the manuscript title (Figshare.com).
Authors’ contributions. S.E.C. carried out the molecular laboratory work,
participated in data and statistical analysis, participated in the design
Goldston and Jennifer Ireland for sample collection of the non-
human primates. We thank Dr Dan Fergus, Dr Holly Menninger
and Dr Greg Pahel for thoughtful discussions. We thank all of
the dedicated citizens who participated in our study and who
contributed samples and ideas for this project.

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