Current Biology

Review

The Molecular Basis of Human Brain Evolution

Wolfgang Enard
Department of Biology II, Ludwig Maximilian University Munich, Grosshaderner Str. 2, D-82152 Martinsried, Germany
Correspondence: enard@bio.lmu.de
http://dx.doi.org/10.1016/j.cub.2016.09.030

Humans are a remarkable species, especially because of the remarkable properties of their brain. Since the
split from the chimpanzee lineage, the human brain has increased three-fold in size and has acquired abilities
for vocal learning, language and intense cooperation. To better understand the molecular basis of these
changes is of great biological and biomedical interest. However, all the about 16 million fixed genetic changes
that occurred during human evolution are fully correlated with all molecular, cellular, anatomical and behav-
ioral changes that occurred during this time. Hence, as humans and chimpanzees cannot be crossed or
genetically manipulated, no direct evidence for linking particular genetic and molecular changes to human
brain evolution can be obtained. Here, I sketch a framework how indirect evidence can be obtained and re-
view findings related to the molecular basis of human cognition, vocal learning and brain size. In particular, I
discuss how a comprehensive comparative approach, leveraging cellular systems and genomic technolo-
gies, could inform the evolution of our brain in the future.

Introduction Independently of the practical limitations to measure pheno-

The evolution of our own species fascinates many of us and is
relevant to understanding our biological and biomedical condi-
tions [1,2]. As our ecological niche is apparently dependent on
special cognitive features, the evolution of our brain is of partic-
ular relevance. The human brain has a long evolutionary history,
but of special interest is the period after we split from our closest
living relatives, the common chimpanzee (Pan troglodytes) and
the bonobo (Pan paniscus). Genetic and phenotypic changes
that occurred after this split and that are common to all currently
living humans can be defined as ‘human-specific’ (Figure 1). In
this review, I will discuss how changes in human brain pheno-
types after the human–chimpanzee split can be linked to
cellular, molecular and genetic changes. To define human-spe-
cific changes, comparisons need to include humans, chimpan-
zees and at least one primate outgroup, such as the orangutan
or the rhesus macaque (Figure 1). Fortunately, the increasing
availability of genome sequences from humans, chimpanzees
and other primates, as well as even our closest extinct relatives,
the Neanderthals and Denisovans [3], allows us to precisely
identify most of the about 16 million genetic changes that
occurred in the human lineage, i.e. after the split from the chim-
panzee lineage and before the most recent common ancestor of
currently living humans (Figure 1, Box 1).
In contrast to these fairly well defined, discrete genetic
changes, the gradual changes in molecular, cellular, anatom-
ical and behavioral phenotypes are much more difficult to reli-
ably measure in a systematic manner, particularly as access
to samples is limited for ethical and practical reasons. Espe-
cially for embryos it is practically and ethically impossible to
obtain invasive samples from chimpanzees and other great
apes. Despite these limitations to comprehensively define
human-specific brain phenotypes across different levels and
during development, substantial progress has been made
in recent years to measure anatomical, cellular and mo-
lecular phenotypes in humans, chimpanzees and other pri-
mates (Box 2).
types, all phenotypic changes and all genetic changes that
occurred before the last common ancestor of currently living hu-
mans are fully correlated (Figure 1). As crosses between humans
and chimpanzees are not an option (see [4] for a historical ac-
count of an attempt in the 1920s), the power of quantitative sys-
tems genetics [5,6] cannot be used to disentangle genotypes and
molecular, cellular and behavioral phenotypes. Furthermore, it is
not possible — again for obvious ethical reasons — to test gene
function by genetically manipulating humans or chimpanzees.
For all of these reasons, direct proof for linking particular ge-
netic changes to particular phenotypic changes in humans
cannot be obtained. In this review, I will sketch a framework of
how plausible indirect evidence can and has been obtained
to tackle this question nevertheless. I will then illustrate this
approach by discussing recent findings related to the evolution
of human social cognition, language and speech as well as brain
size and how the prospective increase of genomic information
and cellular model systems from many primate and mammalian
species should allow a more comprehensive comparative
approach to human brain evolution.
A Framework for Quantitative Systems Genetics without
Crossing
One can view human brain evolution as a problem of doing
quantitative systems genetics with complete access to geno-
types, limited access to phenotypes and no possibility to do
crosses and genetic manipulations. To still reconstruct plau-
sible scenarios of causal relationships between genotypes
and phenotypes during human brain evolution, one needs to
add information from additional sources. This includes the
inference of positive selection on genes or certain regions of
the genome, genotype–phenotype associations within hu-
mans, genotype–phenotype associations within model organ-
isms and genotype–phenotype associations across species.
Such data can — and probably most often must — be com-
plemented with experimental approaches that test particular

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~14 million substitutions
Social cognition
~2 million indels Vocal learning
~24,000 amino acid changes
Brain size
~ 14 million years and ~ 1.5 substitutions/100 bp
genotype–phenotype hypotheses in an experimental system
(Figure 2).
Genotype–phenotype associations within humans are maybe
the most important type of information used to interpret genetic
changes during human evolution. Fortunately, more and more
resources are available that annotate genes and regulatory re-
gions by charting mRNA expression and epigenetic modifica-
tions with high spatial and temporal resolution in the human brain
[7,8]. Furthermore, common and rare genetic variants within
humans can be linked to human phenotypes, including psychiat-
ric diseases, language related phenotypes or brain structures
[9–11]. For model organisms, such as mice, similar resources
exist that complement the human data as genetic and other
experimental manipulations can be done during all stages of
development [12].
While this annotation of the human genome allows re-
searchers to categorize human-specific genetic changes based
on whether they occur in coding regions, putative regulatory
regions or putative non-functional regions, the vast amount of
these changes are thought to be neutral, i.e. having no effect
on fitness or on the brain phenotypes of interest. To identify
genes or regulatory elements that could have been affected by
positive selection and whose alleles could have had a selective
advantage during human evolution, one uses statistical methods
comparing DNA sequence variation within and between humans
and other species [13]. While this is an elegant way to make use
of the available genomic information, it is important to realize that
the power to identify such positively selected elements is low,
especially for genetic changes that are fixed in humans. This is
because a signature of positive selection can only be detected
when either several substitutions in the genetic element were
affected by positive selection or when positive selection on a sin-
gle allele was so recent that the so-called ‘hitchhiking’ effect on
the patterns of variation on the linked genomic regions deviates
significantly from expectations of neutrality. So even when
considering that better primate genome sequences and align-
ments should prevent an excess of false positives that can be
an issue with current genome assemblies [14] and that better
statistical models allow consideration of effects such as biased
gene conversion [15], the false negative rate will remain high at
a given false positive rate.
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Current Biology
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Figure 1. Genetic and phenotypic changes
in the human lineage.
All genetic and phenotypic changes that occurred
Humans
after the split from the chimpanzee lineage and
before the most recent common ancestor of
currently living humans are fully correlated. The
Neanderthal tree is scaled according to divergence times esti-
mated for apes and humans in [111] and [112],
Denisovan respectively. The number of substitutions are
taken from [113], assuming that 80% of all genetic
Chimpanzee changes are fixed in currently living humans [100].
Brain images with permission from [114] and
Bonobo Dean Falk.
Gorilla
Orangutan
In any case, the affected phenotype,
which is the cause of positive selec-
Current Biology
tion, must be inferred from additional
data. Thus, linking positive selection in
genomic elements to human brain evolu-
tion requires additional evidence. This is especially true as evi-
dence for positive selection during human evolution is found in
many protein coding genes but is rarely specific to the human
lineage. The reason for this is unclear, but involvement of genes
in pathogen interactions [16] or non-adaptive processes, such as
compensatory evolution that can lead to positive selection
without changes in function [14,17,18], could be responsible.
Hence, it is crucial to evaluate positive selection occurring on
the human lineage in a comparative context of several primate
or mammalian lineages. A comparative approach has a long
tradition in evolutionary biology for correlating two or more
phenotypic traits across species [19,20] and should be informa-
tive also for correlating genotype–phenotype relationships
across species, as discussed recently in the context of brain
size evolution [21].
While the above sources of information are needed to
constrain hypotheses regarding the genetic and molecular basis
of human brain evolution, they will usually not be sufficient.
Experimental follow-up of hypotheses will be necessary. Which
functional readout is informative will obviously depend on the
context and available knowledge. For example, absorption
spectra of opsin proteins are probably a very informative readout
to infer color vision of mammals [22], but which readouts might
be informative to infer effects on brain size or even cognition is
much less clear. Investigating evolutionary changes in the rele-
vant cell types is important because it is expected that positively
selected genetic variants are specific to particular cell types and
developmental stages in order to avoid negative side effects
of the variant in other contexts [23]. Hence, the possibility to
generate different cell types and brain organoids from induced
pluripotent stem cells is a crucial development to study human
brain evolution, as discussed in more detail below. However,
cellular models are of course limited, especially when complex
behavioural phenotypes are concerned. Although genetically
modified primates are now technically feasible [24], they might
for ethical and financial reasons not be a major option to study
human brain evolution. Hence, mice will remain a major tool
to investigate particular hypotheses on human brain evolution
[25]. However, genome sequencing and genome editing greatly
facilitate experimental investigations also in other organisms and
the contribution of ferrets to mechanisms of brain size evolution
Current Biology

Review

Box1. Genetic changes to the human lineage.

The sequencing of the chimpanzee genome allowed researchers to identify 35 million nucleotide substitutions that differ between
the human reference genome and the chimpanzee reference genome [113]. Half of these occurred in the human lineage and half of
them in the chimpanzee lineage. The sequencing of an outgroup genome, such as that of the orangutan [115] or the rhesus ma-
caque [116] and in particular the resequencing of 79 ape genomes [111], makes it possible to reliably assign single nucleotide
changes to the human lineage. Less than 20% of these are expected to have occurred after the last common ancestor of all
currently living humans [100,113], which means they are still polymorphic in extant humans, resulting in an estimated 14 million
(80% of half of the 35 million nucleotide differences between human and chimpanzee) fixed nucleotide substitutions in the human
lineage (Figure 1). A small proportion of these human-specific changes (31,389 identified according to [3]) occurred after the
split of modern humans from Neanderthals and Denisovans, our closest relatives for which genome sequences are also available,
allowing unique insights into changes specific to modern humans [3]. Over 90% of the genetic changes will not be relevant as less
than 10% of the human genome is evolving under constraint, i.e. is at all functional [99,117]. Changes in the known functional parts
of the genome include about 24,000 fixed encoded amino acid changes as about 60,000 amino acid differences are expected to
occur in 20,000 protein-coding genes between human and chimpanzee [113]. However, protein-coding regions make up only 1%
of the human genome. The remaining less than 9% of functional regions are much more difficult to localize, but they are strongly
enriched in conserved non-coding regions and in regions showing epigenetic signatures of regulatory regions [8,15]. This informa-
tion has also been used to identify functional non-coding regions that are enriched for human-specific substitutions [15], and as the
human genome annotation gets better for non-coding regions, this will allow a more precise annotation of human-specific
changes.
In addition to nucleotide substitutions, humans and chimpanzees differ by about five million insertions and deletions of DNA [113].
While most of them affect just a few base pairs, more than 1000 affect large regions (larger than 2 kilobases) spanning up to
10 million bases [118] totaling about 100 million base pairs of affected sequence between a human and a chimpanzee genome.
Technically, these changes are more difficult to pinpoint, but an analysis of 97 resequenced ape and human genomes allowed
a fairly detailed assessment of rates and events, including about 60 genes that have been affected by deletions and duplications
of exons in the human lineage [118]. While it will be important to increase the quality of primate genomes, especially with respect to
such structurally complex regions [119], genetic information is no longer a limiting factor.
In summary, there are 14 million nucleotide substitutions and about two million insertions and deletions fixed in the human lineage
of which less than 10% will fall into annotated functional regions, including about 24,000 encoded amino acid changes (Figure 1).
Also the vast majority of changes in functional regions are expected to be neutral, i.e. to have no effect on a molecular or organismic
level, and the major problem is to predict which genetic changes might have functional consequences in which phenotypic
context.

and zebra finches to the evolution of vocal learning show the
importance of using a wider array of model organisms to under-
stand human brain evolution.
When investigating human evolution, one possibility is to start
from genetic changes and work one’s way up to phenotypes.
Such a ‘genotype-first’ approach has been suggested to be
feasible for functionally studying the few thousand fixed differ-
ences that have occurred since modern humans split from Nean-
derthals and Denisovans [3]. Another approach is to start from a
phenotype of interest and work down to the cellular, molecular
and genetic changes involved. Both approaches have been use-
ful and need to merge at some point anyway. For the purpose of
this review, I start with phenotypes, such as social cognition,
vocal learning and brain size, and use the framework sketched
above to review relevant recent findings that might help to under-
stand the molecular basis of their evolution.
Cognitive Traits
Many cognitive features have been postulated to be unique to
humans, but only more recently have cognitive phenotypes
been quantified in a comparative context [26,27]. This has led
to important insights, for instance that the ability to understand
others’ inner states and intentions, sometimes referred to as
‘theory of mind’, is not as unique to humans as initially thought
[28]. Of intense current interest is that humans have changed
especially in their social cognition [29], allowing intensive coop-
eration, including morality [30] and in turn cumulative culture [31].
An impressive recent example of comparative psychology in this
context is a study in which proactive prosociality, a key compo-
nent of human cooperation, has been measured in standardized
behavioural experiments across 15 primate species [32]. It was
found that allomaternal care is the best predictor of interspecific
variation, supporting the notion that the emergence of coopera-
tive breeding is an important life-history trait for human brain
evolution.
While the genetics of such phenotypes have not been directly
explored in humans or other primates, it has been claimed that
many of these social cognitive skills (including language) are
impaired in children with autism [33]. Hence, genes and path-
ways associated with autism risk could be candidates for the
evolution of human social cognition. While many genetic variants
have been associated with autism spectrum disorders [34], it
is unclear whether they have evolved differently on the human
lineage. A recent study investigated the protein evolution of
genes associated with different psychiatric disorders across
primates and mammals [35]. While there was ample evidence
for proteins that evolve under positive selection in the human
lineage, positive selection or rate of protein evolution was not
particularly high in the human lineage for either the autism
spectrum disorder-associated genes or any other class of

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Box 2. Phenotypic changes to the human lineage.
Naturally, the vast phenotypic space of human evolution has
been investigated much less systematically than the genome,
but a large number of putative changes on all different pheno-
typic levels has been collected (https://carta.anthropogeny.
org/content/about-moca [120]). Of particular relevance are
increasing amounts of different neuroimaging data in chim-
panzees and rhesus macaques [121,122], neuroanatomical
differences in apes and primates [123] and neuron numbers
in brains across many species [65]. On the molecular level,
important datasets include measurements of protein, mRNA
and chromatin signatures in human and chimpanzee lympho-
blastoid cell lines [124,125] and in dozens of human, chim-
panzee and rhesus brain samples spanning a range of epige-
netic features, including profiling of lipids and metabolites
[126–128]. The availability of individuals or tissue samples is
a major limiting factor for broader and faster phenotyping
(although much better than the limits of accessible pheno-
types from fossil data, which is not covered here). This is espe-
cially true for obtaining comparable samples during prenatal
development, which are for practical and ethical reasons
essentially impossible to obtain from chimpanzees and
many other primates [96].
disease-associated genes. A similar study recently investigated
the protein evolution of ten genes associated with language and
reading impairments in primates, mammals and birds [36]. While
they find evidence for positive selection or accelerated evolution
in the human lineage, including for the well-studied gene FOXP2
and for the dyslexia associated gene KIAA0319, positive selec-
tion is also found outside humans. Another recent study found
that regulatory elements conserved in non-human primates
and evolving faster in the human lineage are enriched in schizo-
phrenia-associated genes [37]. This is a promising approach to
link disease-associated loci to human regulatory evolution. How-
ever, it is currently unclear to what extend this signature is indeed
human-specific, as accelerated evolution of regulatory elements
in other primates has not been investigated. One can expect that
the genetic and molecular mechanisms underlying cognitive
traits and diseases, such as autism, will be much better known
in the near future due to the increasing efforts in human genetics
and genomics [9]. To identify putative genetic changes that
could have been relevant for the evolution of human social cogni-
tion, it will be crucial to use all available information from
comparative data, including traits such as prosociality. If such
genotype–phenotype correlations across species reveal prom-
ising candidates, the validity of phenotypic assays in mice or
cellular systems will probably be the decisive limitation to study
further the genetic basis of cognitive evolution.
Language and Vocal Learning
Language is a highly complex behavior that is certainly unique to
our species and has emerged after the split from chimpanzees
[38]. It is undisputed that the evolution of language has a genetic
basis and that it is helpful to distinguish different physiological,
neurological and cognitive aspects of this complex trait [38,39].
What is disputed intensively is which of these aspects are crucial
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and how they could have evolved. On one end of the spectrum is
the view that language and syntax emerged from the evolution of
more general cognitive specializations, such as shared intention-
ality and sequence learning [40,41]. At the other end of the spec-
trum is the view that a dedicated language module evolved
rapidly, potentially even caused by a single mutation [42]. Lin-
guistic evidence indicates that such a module could have al-
lowed the hierarchical syntactic structure of language by a single
repeatable operation, called ‘merge’, that assembles two syn-
tactic elements a and b to form {a, b} [42]. However, independent
of the neurobiological and genetic plausibility of such a module
emerging suddenly, a phenotype such as the operation ‘merge’
needs to be amenable to empirical investigations at least within
humans before its genetic, molecular or cellular basis can at all
be investigated.
In contrast to the cognitive aspects, the evolution of the main
modality of language, speech, can be studied much better in
a genetic and comparative framework [38]. Speech is a highly
elaborate form of vocal production learning, i.e. the learning of
vocal motor movements based on auditory inputs. In contrast
to communication based on innate vocalizations, vocal learning
is at best very limited in non-human primates, including chim-
panzees [43–45]. However, vocal learning is found in several
other mammalian lineages, including seals, dolphins, elephants
and bats [44], and also evolved independently in the lineages
leading to songbirds, parrots and hummingbirds [46,47]. Two
neuronal pathways, best described for songbirds and parrots,
are crucial for vocal learning in birds: first, the ‘vocal production
pathway’, involving a direct connection from cortical motor areas
to the motor neurons controlling vocalizations; and second, the
‘anterior-forebrain pathway’, a cortico-striatal loop specialized
for vocal learning. In humans, a direct projection from the motor
cortex to the laryngeal motor neurons is necessary to control
vocal production, while monkeys and apes lack such a direct
projection [48]. Similar evidence for a cortico-striatal specializa-
tion in human speech is lacking, but similarities in transcriptional
profiles in birds and humans [49] as well as additional indications
[50,51] suggest that humans might also have evolved a cortico-
striatal loop specialized for vocal learning.
In the search for the genetic basis that gave rise to such adap-
tations, FOXP2 is a strong candidate gene. Humans that are
heterozygous for a non-functional FOXP2 allele suffer from an
impairment that especially affects their speech and language
development [52–55]. Analyses of the evolution of FOXP2
revealed two amino acid substitutions, which became fixed in
the human lineage after its separation from the chimpanzee. It
was suggested that these two substitutions underwent positive
selection, potentially due to their effect on speech and language
[56,57]. Subsequent analyses, including several mammalian
species, suggested that two amino acid changes are indeed
more than expected given the conservation of FOXP2 [36,50].
To test the functional consequences of these two substitutions,
a mouse model carrying these substitutions in the endogenous
FOXP2 gene was generated [58]. These mice showed spe-
cific alterations in the cortico-basal ganglia circuit, including
changes in dopamine levels, striatal synaptic plasticity, dendrite
morphology and gene expression [50,58–60]. Recent evidence
indicates that some of these alterations could be caused by an
increased activity of this humanized FOXP2, leading to increased
Current Biology
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Genome annotations
Comparative approach
GWAS
Mendelian traits
Phenotype –phenotype
Hypothesis on the genetic
basis of an evolved brain
phenoytpe
Genome annotations
QTLs
Experimental testing
knockouts
Human allele Ancestral allele
suppression of the synapse suppressor gene Mef2c and hence
to increased synaptogenesis in the striatum [61]. On a behavioral
level, pups carrying the humanized FOXP2 showed slight but sig-
nificant differences in the pitch of their ultrasonic vocalisations,
but as this was a transient phenotype not found in adults and
as mice do not need a cortex to vocalize normally [62], the
validity of this phenotype for modeling vocal learning evolution
is questionable [63]. However, mice carrying the humanized
FOXP2 allele learned stimulus-response associations faster,
showing that the two amino acid changes do affect striatum-
dependent learning and that such behavioral assays might be
more informative for studying the evolution of human vocal
learning [60]. Importantly, the prediction emerging from these
studies — that FOXP2 affects dopamine-dependent learning
processes in specific regions of the striatum [60] — was recently
supported by findings that dopaminergic innervation changed
specifically in the human lineage and specifically in one striatal
region [64].
Additional genes with different degrees of evidence that they
might be linked to human speech and language evolution have
been found [36,43,49], but also given its history of intense inves-
tigation, FOXP2 is probably still the role model of the possibilities
and difficulties of investigating the genetic basis of the evolution
of a complex human brain phenotype [36].
Brain Size
Maybe the most obvious, best measurable and most studied
aspect of human brain evolution is its increase in size. Only
recently have data been collected to systematically link brain
size to the total number of neurons and non-neuronal cells
across primates, mammals and birds [65]. It could be shown
that in this respect the human brain, including the cognitively
important prefrontal cortex [66], has as many cells as expected
for a primate brain of this size [67]. The human brain is thus a
scaled-up version of a primate brain. The scaling of brain size
and number of neurons can be different in other species such
as rodents and, notably, it was found that elephants possess
fewer cortical neurons than humans [68] and that birds, despite
their small brain size, have neuron numbers similar to primates
[69]. So it is plausible that the total number of cortical neurons
Figure 2. Framework for studying the
molecular basis of human brain evolution.
Studying the molecular basis of human brain
evolution can be regarded as quantitative genetics
without the possibility to cross and genetically
Genotype–phenotype
manipulate the investigated species. Hence,
additional information is required to obtain plau-
sible hypotheses. This includes human variation,
variation in model organisms, variation across
species and evidence for positive selection
or accelerated evolution of genetic elements.
Positive selection Hypotheses usually need to be further tested
Accelerated evolution in experimental systems amenable to genetic
modification such as mice or induced pluripotent
stem cells.
across birds and mammals and their —
Current Biology much less measured — connectivity is
a good predictor of ‘intelligence’ [70].
Therefore, the pure size of our brain with
its 86 billion neurons [67] and probably 100,000 km of connec-
tions [71] might explain a substantial fraction of our cognitive
skills. However, neurons and neuronal activity are energetically
very expensive, using up to 60% of the resting metabolic rate
in human children, and energy uptake needs to be constant
especially during brain development [72–74]. Hence, explaining
how humans can afford such a large brain and still reproduce
more often than smaller-brained apes, such as orangutans, is
a crucial part of understanding human brain evolution. The costs
and benefits of large brains have only recently been analyzed in a
larger multivariate comparative framework and the results indi-
cate that an energetic perspective is crucial to understanding
brain evolution, as lifespan, diet and neonate mass already
explain almost 80% of the variation in relative brain size in non-
human primates [75]. Important aspects as to why humans can
afford even larger brains than other primates could be the inten-
sive cooperative breeding found in humans, their higher amount
of body fat, cooking and the just recently discovered higher
amount of total energy spent by humans compared to apes
[76–78]. It will be interesting to consider such variables when
analysing possible correlations at the molecular and genetic
levels, as soon as genome sequences of more primates become
available [21].
Independent of such ecological and physiological factors and
their underlying molecular processes that allowed humans to
afford large brains, the genetic, molecular and cellular mecha-
nisms that lead to the development of large brains are of intense
current interest. It is beyond the scope of this review to summa-
rize the field, but the crucial point is that the neural progenitor
cells that need to expand in order to build a larger brain have
recently been much better characterized in mice, ferrets, mon-
keys and humans [79,80]. For example, recent experimental
[81–84] and theoretical [85] evidence now strongly suggests
that an expansion of so-called ‘basal progenitor cells’ is suffi-
cient to cause an increase in cortical size and folding that echoes
changes seen during evolutionary increases in cortical size and
folding. As the cellular and developmental basis of brain size
evolution is relatively well understood and as brain size and
correlated variables such as body size, diet and lifespan are fairly
well measured across primates and mammals, the evolution of
Current Biology 26, R1109–R1117, October 24, 2016 R1113

this phenotype is especially amenable to a comparative
approach [21] and other investigations in the sketched frame-
work (Figure 2). This includes experimental approaches to hu-
man brain size evolution that have gained momentum recently.
For example, one study [86] found that the human version of
an enhancer regulating FZD8, a receptor of the Wnt pathway,
leads to a faster cell cycle in neural progenitors and an increased
brain size in transgenic mice, while chimpanzee FZD8 does
not have such an effect. Moreover, another analysis showed
that a human-specific gene duplication encoding Rho GTPase
Activating Protein 11B (ARHGAP11B) increases the proliferation
of basal progenitors when expressed in mice [87]. While these
approaches are crucial first findings, they are certainly not
the only genetic changes responsible for the evolution of
human brain size [88]; thousands of enhancers and promoters
have also gained activity during human corticogenesis com-
pared to rhesus macaques [89]. Furthermore, it is important to
consider that epigenetic signatures and regulatory activity of
particular elements can be different, although the expression
of the regulated gene or network is unchanged. This turn-over
of regulatory evolution is substantial as comparisons of binding
sites between human and mice [90,91] and among mammalian
livers [18] have shown. Also when comparing enhancer signa-
tures and expression levels in human and chimpanzee neural
crest cells, many genes with an increased enhancer signature
in one species are not upregulated on the mRNA level in the
same species [92]. More theoretical, computational and experi-
mental work, including modeling brain size development in
organoids, will be needed to better understand the evolution
of gene regulation in general and during development in
particular [93–97].
Prospects for Studying Human Brain Evolution in a
Comparative Framework
As pointed out above, there has been substantial progress in
describing human brain evolution in recent years. Nevertheless,
we are still far from understanding how the evolution of some
hallmark changes is rooted in genetic, molecular and cellular
processes. The framework of how this could be achieved
(Figure 2) is also helpful to discuss in which areas substantial
progress could be expected in the near future. This certainly
includes increasing knowledge on genotype–phenotype
associations within humans, including rare mutations, as it is ex-
pected that millions of human genomes will be sequenced in the
next ten years [98]. Given a mutation rate of 1–2 3 10–8 per base
pair and generation [99], nucleotide substitutions that occurred
in the human lineage will be mutated back to the chimpanzee
state once in every 1–2 3 108 human chromosomes that are
born [100].
Cheaper and better sequencing technologies will also allow us
to obtain high-quality genomes for essentially all primates and
mammals [101]. This will be important not only to describe pat-
terns of positive selection on the human lineage, but also to
analyze these patterns in a comparative framework to identify
positive selection that co-varies with brain phenotypes of inter-
est, such as brain size or vocal learning. However, to measure
associated molecular and cellular phenotypes in tissues across
many of these species, especially during development, will
remain practically and ethically very difficult. This is to some
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extent similar to the limitations that exist for studying human phe-
notypes, for which induced pluripotent stem cells (iPSCs) bear
great promise to study developmental processes also in com-
plex brain organoids [102]. As iPSCs can be generated from pri-
mates, including apes [103–105], it is evident that they could be a
powerful tool to better understand human brain evolution on a
molecular and cellular level [1,96,97]. Indeed, first comparisons
of differentiated iPSCs from humans and chimpanzees already
indicate that this is the case [92,95]. As molecular phenotypes
such as RNA expression levels can now be measured at the
single-cell resolution [106], this will be a powerful system to anal-
yse the evolution of molecular phenotypes and differentiation
processes in physiologically relevant cell types.
Furthermore, genetic manipulation of primate iPSCs will allow
researchers to probe the genetic basis of brain phenotypes that
can be studied at the cellular level. Massively parallel reporter as-
says now allow us to screen the activity of thousands of en-
hancers [107–109] and genome editing technologies [110] will
enable modification of specific genomic regions in different pri-
mate iPSCs. Together with better theoretical models [93] and
computational tools [94], this will leverage comparative func-
tional genomics to better understand molecular evolution in gen-
eral and human brain evolution in particular.
While iPSCs hold great promise to study human brain evolu-
tion on a molecular and cellular level, they will not abolish the
need to study genetic changes potentially relevant for human
brain evolution in model organisms such as mice. Genome
editing will also make this process decisively faster [96]. While
mouse models will certainly be able to model some aspects of
complex behavioral phenotypes, they will also certainly have
limits [25], eventually restricting which aspects of human brain
evolution we will be able to reconstruct.
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