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THE EVOLUTION OF SEX
DETERMINATION IN ANIMALS
Judith E. Mank1 and Tobias Uller2
1
Department of Genetics, Evolution and Environment, University College London,
London, United Kingdom
2
Department of Zoology, University of Oxford,
Oxford, United Kingdom
INTRODUCTION
Sexual reproduction is the rule in the vast majority of animals, and reproduction by sexual
recombination results in powerful selective forces acting to optimize male and female
phenotypes. Given the ancient origin of the two sexes in the animal kingdom as well as
the selection pressures that hone and maintain them, it would seem reasonable to expect
the genetic pathways underlying sex determination to be highly conserved, especially
given the speed at which deleterious mutations causing intersex or infertile individuals
will be removed from populations. Although there are some examples of conserved sex
determination mechanisms in animals, such as in birds (Mank and Ellegren 2007; Smith
et al. 2009) and therian mammals (Graves 2006), there is overwhelming evidence that
these are the exceptions and that sex determination can be regulated by different genes
even in relatively closely related species. Thus, although male and female sexual phenotypes are conserved, the regulatory control of sex determination is evolutionarily labile
and varies substantially across the animal phylogeny (Gempe and Beye 2010).
Beyond its interest to those studying the evolution of sex, the rapid change in a key
developmental pathway makes sex determination an excellent model for studying the
Advances in Evolutionary Developmental Biology, First Edition. Edited by J. Todd Streelman.
© 2014 John Wiley & Sons, Inc. Published 2014 by John Wiley & Sons, Inc.
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The Evolution of Sex Determination in Animals
evolution of genetic networks (Davidson 2006; Wilkins 2002). The strong selection against
intersex individuals suggests that selection should have optimized developmental signals
regarding sex, canalizing them to be released at the correct developmental time. The outstanding problem is then to understand how changes in the genes underlying maleness and
femaleness arise without causing disruptions to the sex determination pathway, as these
disruptions would lead to the production of suboptimal male and female phenotypes and
purifying selection acting against them acting to preserve the status quo.
Despite the essential nature of sex determination, we understand the genetics of it in
surprisingly few animals. Those animals for which the genetics are relatively well characterized tend to rely on a network with a locus of major effect (the “master sex regulator”;
some of which are shown in Table 2.1). This also has the interesting consequence that the
genetic complement is determined at fertilization and, thus, that sex is often inherited in
a simple Mendelian fashion. However, although they are the best characterized, mechanisms of genetic sex determination (GSD) involving a single locus of major effect are by
no means predominant in animals. There is increasingly strong evidence for polygenic sex
determination in fish (e.g., Bradley et al. 2011; Cnaani et al. 2008; Shirak et al. 2006),
many species have environmental sex determination (ESD) (Sarre et al. 2011), some have
complex relationships between polygenic and environmental factors (Quinn et al. 2007;
Radder et al. 2008), and many others can alter their sex over their lifetime in response to
ecological or demographic conditions (Avise and Mank 2009). What little is known about
the evolution of sex determination has been reviewed numerous times (recent examples
include Gempe and Beye 2010; Graves 2006; Heimpel and de Boer 2008; Schutt and
Nothiger 2000; Williams and Carroll 2009), and rather than reiterate this material, we will
only briefly paraphrase it and draw attention instead to how transitions in the sexdetermining system is expected to be reflected at the level of gene networks, how changes
in this network have important evolutionary implications, and how we might best go
forward to study these aspects of the evolution of sex-determining systems.
EVO-DEVO OF SEX DETERMINATION
Although we think of the sexes as discrete phenotypes, male and female differences
emerge from an undifferentiated embryo at some point during development. Additionally,
although female and male sex differences are most obvious in adults, these dimorphisms
begin as small differences early in development and amplify as the individual matures
(Mank et al. 2010). Sex determination is therefore perhaps best defined as the processes
that underlie differentiation of key components of the sexual phenotype during ontogeny
(Uller and Helantera 2011).
At the most basic level, this differentiation revolves around the formation of testes
versus ovaries, but sexual differentiation can also involve somatic tissue into distinct male
and female types. Under this definition, a sex-determining system represents a particular
structure of the developmental regulation of this process, such as the master trigger system
found in most mammals. In therian mammals, sex determination is a highly canalized
process where expression of a single genetic element early in gonad differentiation (the
SRY gene) is sufficient to cause development of testes (Koopman et al. 1991; Sinclair
et al. 1990). In other specieswhere sex is based on genotype, sex-determining systems
show little genetic variation and constitute a discrete form of phenotypic plasticity in which
environmental conditions experienced at some point during development regulate the
expression of maleness or femaleness via a developmental switch.
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Females: XX
Males: XY
Females: Heterozygous at CSD
Males: Homozygous at CSD
Females: Diploid, with paternal
allocation of Nvtra
Males: Haploid, without
paternally imprinted allocation
Hermaphrodites: XX
Males: XO
Variable
Females: XX
Males: XY
Females: ZW
Males: ZZ
Drosophila melanogaster
Nasonia vitripennis
Caenorhabditis elegans
Musca domestica
Therian mammals
Birds
Apis mellifera
Sex Determination
Clade or Species
TABLE 2.1. Key Positions in Animal Sex-Determining Networks
X chromosome : autosome ratio (I) initiates sex-specific splicing
of Sxl (II) that is maintained via a positive feedback (III)
Heterozygosity versus homozygosity/hemizygosity at the csd locus
(II) results in different expression of downstream elements (III)
whose expression is maintained via positive feedback involving
alternative splicing.
Female-specific splicing of Nvtra (II) is initiated by paternal
allocation of Nvtra mRNA in eggs (regulated by unknown genes;
I) and expression of zygotic Nvtra regulated by imprinted genes
(I), which initiates and maintains downstream sex-specific gene
expression through self-regulatory loops (III)
X chromosome: autosome ratio (I) results in dosage-dependent
expression of XO-lethal 1 (xol-1). In XX hermaphrodites, xol-1
(II) is repressed, leading to expression of tra-1 (III). X : A ratio
of 0.5 in males leads to expression of xol-1, in turn inhibiting
tra-1.
Sex-specific splicing of Mdtra occupies a central node (II) in the
sex-determining network. Depending on the population or strain,
sex-specific Mdtra expression can be initiated by the presence of
one or several Y-linked or autosomal genes and maternally
transferred Mdtra mRNA (I and II). Once initiated, sex-specific
splicing of Mdtra is subject to positive feedback supported by
additional genetic elements (III).
Autosomal genes (I) trigger expression of a Y-linked element only
present in males (Sry, II) that in turn triggers expression of a
non-sex-linked gene (Sox9, II) whose expression is being
maintained via a positive feedback involving several loci (III)
Dosage mechanism on the Z chromosome results in higher
expression of Dmrt1 (I).
Mechanism of Action (Roman Numerals Refer to Figure 2.2,
When Details Are Known)
Smith et al. (2009)
Koopman et al.
(1991); Sinclair
et al. (1990)
Hediger et al. (2010)
Zarkower (2001)
Verhulst et al. (2010)
Gempe et al. (2009);
Hasselmann et al.
(2008)
Bopp et al. (1991)
Key Paper or Recent
Review
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The Evolution of Sex Determination in Animals
Although we understand several GSD networks in detail, we know very little about
ESD networks. It is therefore not clear whether GSD and ESD employ the same underlying pathways with different regulatory elements (GSD would be controlled by a constitutive promoter, ESD by an inducible one), or if the underlying network structure for these
two types of sex determination are completely different. Given the speed of transitions
between ESD and GSD (Bull 1983; Mank et al. 2005; Pokorná and Kratochvíl 2009), and
systems involving both genetic and environmental effects in some organisms (Quinn
et al. 2007; Radder et al. 2008), it seems unlikely that the entire network differs in fundamental ways between ESD and GSD, as there is simply not enough evolutionary space
separating these states in many phylogenies to allow for complete network turnover.
However, there is no definitive information to support or refute this.
Some of the key problems for the evo-devo of sex determination is therefore to understand how the developmental networks of sex determination become structured in particular ways in the first place (e.g., the master trigger systems), how novel determinants of
sex arise within or outside of existing networks, and the implications this has for the rate
and pattern of evolutionary diversification of sex-determining systems.
THE ORIGIN OF NETWORK NOVELTY
Before we discuss the evolution of sex determination networks in particular, it is perhaps
useful to discuss some basic predictions regarding the evolution of genetic networks in
general. Both the elements within the network and the structure of the networks themselves
can diverge and change over time (reviewed in Davidson 2006; Gompel and Prud’homme
2009; Stern 2010), and recent comparative evidence suggests that some parts of genetic
networks are hotspots for evolutionary change, and are therefore more likely than others
to diverge over time.
First, genes with higher mutation rates or with a broader mutational target (i.e., the proportions of mutations that have functional consequences) should more often contribute to the
origin of novel variation in networks and therefore form the basis of network change. This
prediction is based on the need for genetic variation for evolutionary change. Genes with few
tolerable mutations are too constrained to contribute to evolvability, and loci where alleles
have little to no phenotypic effect are invisible to selection and are therefore unlikely to contribute to evolutionary change. It is important to note, however, that the effect of a single locus
is expected to depend on the genetic background (e.g., Nijhout and Paulsen 1997).
Second, changes in genes that occupy the central nodes in networks, and which control
the expression of many downstream genes, can typically generate phenotypic change via
fewer mutational steps than changes in structural genes. For example, it has been suggested
that the evolutionary lability of the dorsal and ventral trichome pattern in Drosophila
larvae has been facilitated by the central position of the gene shavenbaby, which acts as
a node as it integrates several inputs and triggers expression of a constellation of downstream genes that contribute to cell differentiation (Stern and Orgogozo 2009). Therefore,
the network position and connectivity of this gene make it a particularly likely target for
evolutionary changes in trichome patterning.
Third, network evolution can operate from changes and refinements of regulatory
mechanisms. There are at least two routes to this type of innovation. First, the origin
and fixation of fine-scale regulatory patterning via duplication and specialization of
cis-regulatory elements (Carroll 2008) allows for localized expression of previously
broadly expressed genes. Fine-scale expression partitioning due to cis-regulatory additions
Evolution of Genotypic Sex Determination 19
maintains the network position of a gene while reducing potential negative pleiotropic
effects in other parts of the body that would result in loss via purifying selection. Alternatively, gene duplication allows for subfunctionalization or partitioning among the daughter loci of the function once completely controlled by the parental gene. This can resolve
pleiotropic constraints, particularly related to sex determination and sexual phenotypes
(Gallach and Betran 2011; Gallach et al. 2010).
Beyond these three predictors, evolutionary shifts in the developmental regulation of
sex determination will also depend on population-level processes (Snell-Rood et al. 2010;
Stern 2010). For example, mutations with more dramatic effects on the phenotype may be
more likely to go to fixation in small populations. Small populations are also more subject
to drift and have lower number of mutations introduced per generation (Hartl and Clark
1997), which may result in selective fixation of genetic variants that determine sex in a
less-than-optimal way. The strength of selection will also contribute to the rate of evolutionary change and may influence the positions in gene networks that are most likely to change;
strong selection makes it more likely that genes of major effect go to fixation. Relaxation
of selection, on the other hand, allows accumulation of mutations and can facilitate divergence in regulatory networks, perhaps in particular in parts of the network that are subject
to low levels of pleiotropy and high rates of mutation (Snell-Rood et al. 2010).
EVOLUTION OF GENOTYPIC SEX DETERMINATION
A series of now classic scientific discoveries in the early 20th century established that sex
in some animals, both invertebrate and vertebrate, followed Mendelian inheritance based
on presence or absence of particular chromosomes (reviewed in Maienschein 1984). This
established sex as a genetically determined character, inherited as a dominant or recessive
trait. GSD, often related to sex chromosomes, has remained the dominant model of sex
determination, although it is clear that it may not be the most common.
The Developmental Basis of GSD
In line with expectations based on models of adaptive evolution (Box 2.1), GSD often
involves a single locus of major effect. The genetic networks that have been described in
greatest detail are those of the insect Drosophila melanogaster, the nematode Caenorhabditis elegans, and the mammal Mus musculus, and there are excellent and profuse reviews
Box 2.1. Adaptive Evolution of GSD and ESD
Although it is possible that some sex-determining systems are selectively neutral,
the phylogenetic pattern of GSD and ESD suggest a selective explanation for their
diversity. Because sex determination has a direct impact on the sex ratio, sex ratio
selection is expected to strongly influence evolution of sex-determining systems
(review in Uller et al. 2007). For example, frequency-dependent selection against
the most common sex will tend to favor evolution of equal sex ratios, more formally
expressed as the equal investment in sons and daughters (e.g., Pen and Weissing
2002; West 2009). This could select against environmental effects on sex determination. Furthermore, although polymorphisms can be determined by genetic variation at several different loci, systems with only two morphs tend to favor evolution
of a single locus of major effect (Bull 1983; Kopp and Hermisson 2006; Rice 1986).
(Continued)
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The Evolution of Sex Determination in Animals
The reason for this is that a single-locus genetic architecture allows optimal “matching” of the morphs to the selective conditions that arise from intraspecific competition (Kopp and Hermisson 2006). Thus, although complex multilocus GSD systems
have been shown to exist (Ser et al. 2010; von Hofsten and Olsson 2005), polygenic
sex determination may be unstable and evolve toward a system with a gene of
major effect, although it does not necessarily predict whether the system will
exhibit male or female heterogamety. This locus does not even need to be expressed
in the embryo; GSD via maternally expressed genes are also possible (Kozielska et
al. 2006; Werren et al. 2002), and the evolutionary outcome may be sensitive to
the extent of sexual and parent–offspring conflict (MacCarthy et al. 2010; van
Doorn and Kirkpatrick 2007).
Although sex ratio selection means that there will always be an element of
frequency-dependent selection, theoretical models have shown that ESD should be
favored when one sex benefits more than the other from the environment during
development, or if the environment correlates with sex-specific selection at some
point later during ontogeny (Shine 1999). The adaptive significance of ESD is easy
to understand in the case of social sex determination in some species of invertebrates and fish. For example, size-advantage models may explain the origin of
sequential hermaphrodites, where reproductive fitness is greater for larger animals
of one sex and smaller animals of the other. For species with indeterminate growth,
this suggests that most adult individuals will start out as one sex and reach a size
threshold at which reproductive fitness is maximized by changing to the other sex
(Charnov 1982). In protandrous species, the cheaper cost of sperm means that an
individual’s lifetime fitness is maximized by starting adulthood as a male, and only
changing to a female when of sufficient size to bear the cost of egg production.
In other species with male competition based on size, male reproductive fitness is
minimal until a size threshold is crossed, and it is therefore advantageous to start
as a female.
In vertebrates, the most common gonochorist ESD is temperature-dependent sex
determination, or TSD (Valenzuela and Lance 2005). The adaptive significance of
TSD has been elusive, but studies of fish and lizards suggest that one possibility is
that the temperature during development influences developmental time and
therefore correlates with the timing of hatching or birth, which may have sexspecific fitness consequences (Conover 1984; Pen et al. 2010; Warner et al. 2009).
If timing of hatching is the key fitness characteristic, this suggests that other environmental factors that also affect developmental time may influence sex determination in TSD species. This is consistent with emerging evidence that although
temperature has strong effects in the laboratory, its effect can be less straightforward under natural conditions (Warner and Shine 2011). However, a direct link with
developmental rate has not yet been demonstrated. Nevertheless, recent theoretical modeling suggest that sex-determining systems that are highly canalized toward
being responsive only to temperature may be suboptimal and that mechanisms that
integrate several different sources of environmental input into sex determination
should be favored by selection (Schwanz et al. 2010).
on the exact mechanisms of sex determination in all these animals (e.g., see Brennan and
Capel 2004; Gempe and Beye 2010; Stothard and Pilgrim 2003). We therefore limit our
discussion to the key elements that define the structure of the genetic networks in order
to provide a baseline for the comparative evidence from species with less detailed
information.
In Drosophila, which is male heterogametic and has a male-limited Y chromosome,
sex determination is based on the X : A ratio early in development (Figure 2.1A) before
Evolution of Genotypic Sex Determination A
Male
21
Female
Karyotype
X
Ratioof X chromosomes
to autosomes
Y
X : A = 1
Sxl
Sxl
tra + tra‐2
tra + tra‐2
dsxM
B
X
X : A = ½
Genetic pathway
Phenotypes
X
fruM
Male
Male
morphology behavior
dsxF
dsxM
Female
morphology
Male
Female
X Y
X X
Karyotype
Genetic pathway
Sxl + Male factor
Sxl
tra + tra‐2
tra + tra‐2
dsxM
Phenotypes
Male
morphology
dsxF
dsxF
dsxM
Female
morphology
Figure 2.1. Comparison of sex determination in the Dipterans, Drosophila melanogaster, and
Ceratitis capitata. Panel A: Sex determination in Drosophila is based on the ratio of X chromosomes to autosomes. The X chromosome to autosome ratio functions when multiple X-linked
transcription factors bind to and activate the sex lethal (Sxl) gene, and the activated Sxl product
then initiates sex-specific splicing of transformer (tra) mRNA, and indirectly of the doublesex
gene (dsx). This pathway leads to alternative splicing of dsx, with the male form (dsxM) ultimately
leading to the development of a testis, and the female variant (dsxF) leading to an ovary. Both
variants of dsx involve exons 1, 2 and 3, with dsxM including exons 5 and 6 and dsxf ending in
exon 4. Panel B: In Ceratisis, sex determination is not based on X : A ratio, rather a Y-linked
element that represses tra and tra-2, ultimately leading to sex-specific splicing of dsx and male
and female phenotypes.
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The Evolution of Sex Determination in Animals
the onset of sex chromosome dosage compensation (Bopp et al. 1991). This dosagesensitive mechanism functions when multiple X-linked transcription factors bind to and
activate the Sxl gene, and the activated Sxl product then initiates sex-specific splicing of
transformer (tra) mRNA, and indirectly of the doublesex gene (dsx). Importantly, once
this process is initiated, feedback loops ensure continued expression of Sxl and maintains
female differentiation. The Drosophila system is therefore composed of a key genetic
element, Sxl, that occupy a node in the sex-determining network, but where several X
chromosome genes regulate the expression of Sxl itself.
Sex determination in C. elegans shares many elements with Drosophila, both in terms
of the X chromosome dosage that initiates regulation of the gene xol-1, as well as conserved genes such as tra (Conradt and Horvitz 1999). However, sex determination in C.
elegans does not seem to involve sex-specific splicing, which thus far appears to be limited
to the insects. Finally, C. elegans is interesting in that XX individuals develop as hermaphrodites, which sequentially produce first male, then female gametes. XO individuals
develop as males. Androdioecious mating systems such as this, though common in plants,
are relatively rare in animals, and may prevent the spread of a dominant male-determining
factor such as that seen in mice (see later) as it would preclude the development of hermaphrodites. It is worth noting that the androdioecy in C. elegans is likely a recently
derived state from within a dioecious clade, where individuals are either male or female
and do not switch sex.
Sex determination in mice is not based on dosage; rather, the gene that occupies the
central point in the network (Sry) is present only in the male as it located on the Y chromosome. It is not clear whether a single or multiple factors regulate the expression of Sry
(Sekido and Lovell-Badge 2008). However, it seems like the direct target of the Sry protein
(SRY) is a single gene, Sox9, which itself is located on an autosome. Sox9 is necessary
for testes formation in the mouse and, after the initial initiation of its transcription by Sry,
its expression is maintained assisted by other genes and feedback loops. Failure to maintain
SOX9 can lead to development of ovaries in XY individuals with a functional Sry gene.
Indeed, unless SOX9 reaches the threshold that initiates the feedback loop that maintains
its expression, accumulation of β-catenin in the genital ridge suppresses SOX9.
This brief overview emphasizes some key aspects of the regulation of genotypic sex
determination that are important for understanding evolutionary transitions between sexdetermining systems (Figure 2.2). First, all networks have at least one central node, the
genetic element often referred to as a master trigger. Second, at least in D. melanogaster
and C. elegans, this gene also resides in a convergence point in the gonad differentiation
network, where it integrates several different inputs into a single signal that in turn
switches on multiple other genes. Third, the gene of major effect may or may not be
inherited through only one sex. Fourth, once initiated, maintenance of the expression of
key elements requires feedback systems, and the elements involved in this feedback system
are crucial for the master trigger to have a major effect on sex determination.
Evolutionary Transitions between Genotypic Sex-Determining
Systems
Are the comparative patterns of sex determination consistent with the hypotheses for
where in genetic networks evolutionary change is most likely to occur? More is known
about the diversity of sex determination in insects than in any other taxa, making them
the obvious candidates for investigating this question. The comparative evidence so far
Evolution of Genotypic Sex Determination I. Non-sex-specific elements (e.g.,
transcription factors)
II. Master trigger (e.g., SRY) and
down stream sex-specific
expression (e.g. SOX9)
III. Down stream sex-specific
expression (e.g., activation and
suppression)
Figure 2.2. Generalized model of a sex determination network based on current understanding
from GSD species. The GSD network often is located at the convergence of the gonadal differentiation network, which integrates several different inputs (maternal, environmental, genetic;
Panel I) into a single element or multiple hierarchically ordered elements occupying a central
node in the network (Panel II). The central node then in turn switches on multiple other genes.
Once initiated, maintenance of the expression of downstream key elements requires feedback
systems and the elements involved in this feedback system are crucial for the central element to
have the expected major effect on sex determination (i.e., to be a master trigger). Novel sexdetermining factors could arise from any part of the network or even from the outside as long
as it influences the upstream initiation or downstream maintenance of expression of key elements that contribute to development of testes or ovaries. See Table 2.1 for examples of genes
positioned at the three parts of the network in different species.
suggests that many of the systems of sex determination in insects are based on a similarly
shared mechanism of sex-specific splicing of the transformer mRNA (Gempe and Beye
2010), where one form leads to female development and the other to male development.
More importantly, sex-specific splicing of a single-gene downstream on the sex determination pathway (tra) is conserved in insects (Gempe et al. 2009; Lagos et al. 2007; Pane
et al. 2002; Pomiankowski et al. 2004; Raymond et al. 1998; Verhulst et al. 2010; Williams
and Carroll 2009) and is even involved in sex determination in C. elegans (Conradt and
Horvitz 1999; Goodwin et al. 1993).
Although other downstream elements are conserved in addition to tra (Marin and
Baker 1998), this conservation is not present in the integrative control point or node of
the network, and there are many different regulatory mechanisms that initiate the sexspecific splicing that ultimately leads to sex determination. This is consistent with the
general prediction that nodes in genetic networks are hotspots for evolutionary innovation
because they allow change without disruption of development. However, it does not necessarily imply that sex-determining networks evolve simply by mutations in existing nodes
or upstream addition of master triggers. Comparisons between Drosophila and the medfly,
Ceratitis capitata, are particular interesting (Figure 2.1) as they show how the downstream
mechanisms can be retained despite substantial changes upstream, even involving transitions between dosage effects and Y-linked master triggers. Specifically, in contrast to
Drosophila, Sxl is not expressed in a sex-specific manner in medflies. Instead, current
evidence points toward regulation of the alternative slicing of the ortholog of tra via a
Y-linked element (Saccone et al. 2002). The alternative splicing of tra is maintained via
positive feedback once initiated in the early zygote. That dramatic changes at the level of
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The Evolution of Sex Determination in Animals
sex-determining systems can arise from very simple gene regulatory interactions is also
supported by theoretical models (MacCarthy et al. 2010).
Although the available evidence supports that evolutionary transitions between GSD
systems occur relatively upstream in sex-determining networks, the origin of recruited
genes is not well understood. The prediction that genes where mutations are tolerable and
which have functional phenotypic consequences (i.e., avoid disruption of development by
virtue of low pleiotropy) should be more likely to emerge as master triggers has little
direct empirical evidence in terms of sex determination. No one has, to our knowledge,
done a study on mutability and phenotypic effects of sex-determining candidate genes in
a systematic way that would be required to test this prediction. However, analyses of the
transcription network in crosses between mouse strains that differ in their susceptibility
to sex reversal (relative to the presence of Sry) have shown that autosomal regulatory
elements that are involved in maintaining SOX9 expression or the Wnt/β-catenin signaling could be a rich source of genetic variation for evolutionary shifts in GSD networks
(Munger et al. 2009). This means that rather than the novel master trigger being a novel
genetic element added upstream to the sex-determining network (Wilkins 1995), such
genes of major effect may be recruited from downstream genes involved in maintaining
SOX9 expression, resulting in a neo-sex chromosome. Alternatively, Sox9 may show sufficient genetic variation in its sensitivity to X-linked transcription factors (or those factors
may show genetic variation themselves) to allow evolution of a dosage system analogous
to that of Drosophila. Further studies of rare incidence of evolutionary loss of Sry in
mammals (Just et al. 1995) are likely to be informative with respect to the extent of
change in sex-determining networks after random or selective loss of existing master
triggers.
Evidence from vertebrates suggest that network novelties do not always arise directly
from variation in genes within the network, but are instead duplicates of loci already
present in the sex determination pathway. An indicative example of this comes from the
medaka rice fishes in the genus Oryzias. Within this clade, a duplicate of the Dmrt1 gene,
called Dmy, has taken over as the master regulator of male sex determination along one
lineage (Matsuda et al. 2002; Nanda et al. 2002). Dmrt1 has a conserved role in sex determination spanning invertebrates and vertebrates (Matsuda et al. 2002; Smith et al. 2009;
Yi and Zarkower 1999; Yoshimoto et al. 2008) and therefore Dmy was, at the time of
duplication, already well configured to play a role in this pathway. It is yet unknown to
what extent such changes in genes of main effect in vertebrates was initiated by the gene
duplication and whether additional changes are also involved in the regulation of DMY
expression or maintenance of downstream feedback loops.
In conclusion, in both insects and vertebrates, there seems to be substantial evolutionary lability, but primarily at the top of the sex determination pathway. Additionally, in
vertebrates at least, this change seems to be due to changes in a limited repertoire of genes
that are already implicated in sex determination, with paralogues often emerging as master
regulators. This sort of recycling within a framework of conserved downstream genes may
explain how transitions among GSD mechanisms occur without disrupting the development of sex-specific phenotypes. Nevertheless, the conclusions that can be drawn from
this observation are limited because the majority of studies on nonmodel organisms are
limited to the study of homologues of genes found to play a role in sex determination in
mice and humans. Indeed, recent evidence from cichlids suggests that novel main elements
in sex-determining networks can originate from genes ancestrally involved in differentiation of secondary sexual characters (Roberts et al. 2009; Ser et al. 2010). Disentangling
the mechanistic basis of this acquired function will help to test predictions regarding the
Evolution of Genotypic Sex Determination origin of network novelty, in particular whether pleiotropy facilitates or constrains evolution of novel sex-determining genes.
GSD and the Evolution of Sex Chromosomes
Most of the models for studying sex determination possess highly differentiated sex chromosomes, such the X and Y chromosomes seen in therian mammals, Drosophila, and C.
elegans, and the Z and W chromosomes in birds. This is not to say that distinct sex chromosomes always, or even usually, co-occur with GSD, but that some systems with GSD
may eventually present them. It is important to point out that highly differentiated sex
chromosomes are not inevitable, as old but undifferentiated sex chromosomes exist in
ratite birds (Mank and Ellegren 2007) and snakes (Matsubara et al. 2006). When they do
occur, the evolution of highly differentiated sex chromosomes may act as a brake on further
changes to the sex-determining pathway. Circumstantial evidence for this comes from the
fact that clades with old heteromorphic sex chromosomes, such as birds, mammals, and
Drosophila, show little evidence of change in sex determination.
There are several reasons why this might be the case, many relating to the unusual
sex-specific selection pressures acting on sex chromosomes. Very briefly, sex chromosome
systems come in two major flavors: female heterogamety where females have one Z and
one W and males have two Z chromosomes, and male heterogamety where females have
two X chromosomes and males one X and one Y. The W and Y chromosomes are roughly
analogous to each other in that they are both sex-limited, with the W present only in
females and the Y only in males. The type of sex chromosome may depend on the type
of sex determination, with recessive male mechanisms or dominant female master regulators resulting in ZZ-ZW systems, and dominant male master regulators and recessive
female mechanisms in XX-XY system.
The divergence of X-Y and Z-W sex chromosome pairs is based on a model of sexually antagonistic alleles for genes near the sex-determining locus (Charlesworth 1991; van
Doorn and Kirkpatrick 2007) that results in selection for recombination suppression
between the sex chromosomes and therefore allowing them to diverge from one another.
Sexually antagonistic alleles benefit one sex at a cost to the other, and if sex chromosome
differentiation is the result of the accumulation of sexually antagonistic alleles, highly
differentiated sex chromosomes should have large numbers of antagonistic loci. Because
changes in the master regulator often cause sex chromosome loss or turnover (Kitano et
al. 2009; Nanda et al. 2002; Ross et al. 2009), changes in GSD may be difficult for lineages
with highly differentiated sex chromosomes because loss of those sex chromosomes will
result in the loss of a linkage group that carries several loci with sex-specific effects,
thereby resulting in less fit individuals. It is important to point out here that there is no
strong empirical evidence for this model of sex chromosome evolution (Ironside 2010).
Additionally, aside from sex-determining genes themselves, there are no known examples
of sexually antagonistic loci where the cost to one sex and the benefit to the other has been
measured. This means that although there are indications that large amounts of intralocus
sexual conflict are resident within genomes (Bonduriansky and Chenoweth 2009; Harano
et al. 2010; Lewis et al. 2011), no one really knows at the molecular or biochemical level
what a sexually antagonistic allele actually looks like.
Even if this model of sex chromosome differentiation is not common, highly differentiated sex chromosomes, particularly the W and Y chromosomes, have important sexspecific functions in fertility and mate choice (Hori et al. 2000; Lange et al. 2009; Lemos
et al. 2008; Moghadam et al. 2012; Postma et al. 2011), and this alone may present a
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The Evolution of Sex Determination in Animals
serious barrier to change in GSD as males lacking a Y chromosome or females lacking a
W would be infertile or subfertile and therefore removed from the population by negative
selection.
EVOLUTION OF ENVIRONMENT-DEPENDENT SEX DETERMINATION
Environment-dependent sex determination is a polyphenism, that is, a form of phenotypic
plasticity with discrete phenotypes. Polyphenisms are common in nature, in particular in
invertebrates. Familiar examples include queen and worker morphs in social insects,
density-induced winged morphs in aphids, and seasonal morphs with different colours and
patterning in some species of butterflies (reviewed in West-Eberhard 2003; Whitman and
Ananthakrishnan 2009; Simpson et al. 2011).
Polyphenism describes the sexes in both gonochorist ESD lineages, where sex is
determined by some environmental cue or cues and is maintained throughout the remaining life span, and sequential hermaphrodites that change sex based on some ecological
factor. The former are illustrated by the temperature-dependent sex-determining mechanisms in turtles and crocodilians (Valenzuela and Lance 2005), and the latter are exemplified by the protandous (male then female) or protogynous species of many fish (Avise and
Mank 2009).
The Developmental Basis of ESD
The developmental basis of ESD is poorly understood compared to that of GSD systems,
largely because no model organisms have ESD. The best studied examples so far are
temperature-dependent sex determination (TSD) in reptiles and fish (Valenzuela 2008;
Valenzuela and Shikano 2007) and the protogynous gobies (Black et al. 2005). Studies of
the developmental basis of nonsex polyphenisms have revealed that they typically are
hormonally regulated (Nijhout 1999, 2003), suggesting that this may also be the case for
sex determination. Indeed, with the exception of placental mammals and birds, both ESD
and GSD vertebrates can be “sex-reversed” by application of estrogens or estrogeninhibitors, showing that some aspect of gonad differentiation is estrogen dependent (e.g.,
(Freedberg et al. 2006; Shine et al. 2007). There is also some evidence that other hormones
(androgens and corticosterones) can influence sex determination (Hattori et al. 2009), but
whether they form a part of a normal, species-typical, sex-determining developmental
network remains unclear. Nevertheless, a hormonal basis for ESD systems is well supported, at least in vertebrates.
Despite the fundamental role of estradiol in sex determination in ESD species, very
little is known about the molecular mechanism by which the environment exercises its
effects, or how estradiol influences gonad differentiation (e.g., what genes it upregulates).
Studies of reptiles and fish have shown that orthologues of many of the genes that are
involved in sex determination in mammals and birds are expressed in gonads of TSD
species (see table 1 in (Rhen and Schroeder 2010). However, the timing of expression
and the extent to which it is sex-specific varies between genes and between species. For
example, although SOX9 and DMRT1 are relatively consistently expressed differently in
gonads developing at male- and female-producing temperatures, expression of estrogen
receptor α and SF-1 are less consistent (Rhen et al. 2011). Only recently have the temperature- and estradiol-sensitivity of those genes started to be elucidated. For example,
in painted turtles, estradiol suppresses SOX9 expression during the latter phase of gonad
Evolution of Environment-Dependent Sex Determination differentiation (Barske and Capel 2010). This suggests that estradiol primarily functions
as part of the feedback loop that maintain SOX9 expression, but that it is of limited
importance in the early stages of gonad differentiation. Recent evidence from isolated
cultured gonads of the painted turtle also shows that several orthologues of genes
involved in sex determination in mammals show temperature-dependence in their expression levels (Shoemaker-Daly et al. 2010). This may point toward a regulatory network
in which several different genes show temperature dependence in their expression levels.
Evolution of some level of redundancy, with several genes that show the same
environment-dependent expression, may also be selectively favored because it increases
the potential for divergence of developmental trajectories and reduces the risk of intersexes. If this is the case, then the main effect of temperature is better thought of as a
mechanism that sustains differential gene expression that results in cumulative differences in gonad differentiation rather than as a master trigger of sex determination (Uller
and Helantera 2011). However, in other species, the window of temperature sensitivity
is narrow and occurs very early in development (even before gonad differentiation;
reviewed in Baroiller et al. 2009). In these species, temperature may either trigger
expression of an upstream gene that subsequently switches on a battery of downstream
genes, analogous to the GSD systems described earlier, or trigger epigenetic changes to
genes involved in initiation or maintenance of the estrogen-dependent feedback loop that
sustains gonad differentiation.
Support for the latter scenario comes from recent research on seabass. In this species,
high temperature before the onset of gonad differentiation increases DNA methylation in
the promoter of the gonadal aromatase gene (cyp191a), which results in suppression of
transcription and hence a reduction in the expression of aromatase in the gonads, leading
to the development of testes (Navarro-Martín et al. 2011). Similar mechanisms may be
important in other species with narrow, early windows of thermal effects on sex determination, whereas those where sustained temperature during gonad differentiation is needed
may have different means of regulation of aromatase activity.
Evolutionary Transitions between Environment-Dependent
Sex-Determining Systems
The comparative pattern of ESD and its developmental basis is still too poorly understood
to address what is the developmental basis of the transition between different mechanisms
of ESD systems. However, we can identify two different kinds of transition. First, changes
in the genetic regulation of ESD, while retaining sensitivity to the same environmental
input (Nahmad et al. 2008; True and Haig 2001) and, second, changes in the use of cues
with more or less conserved regulation of the genes involved in sex determination
(Schwander and Leimar 2011).
Examples of the first kind could be sought by comparing TSD species with different
windows of temperature sensitivity (Valenzuela 2008, 2010). For example, we predict that
in species where sex is sensitive to temperature throughout a substantial part of gonad
differentiation, the network will essentially consist of downstream elements (i.e., the
feedback loop in Figure 2.2), with the expression of one or more key genes being directly
influenced by temperature. Evolutionary shifts to successively earlier and more narrow
windows of temperature sensitivity should be accompanied by upstream addition of
genetic elements that regulates SOX9 expression or, alternatively, stable epigenetic modification of key elements involved in sustaining, for example, aromatase activity later in
the developing gonad.
27
28
The Evolution of Sex Determination in Animals
Promising candidates for studying evolutionary shifts between reliance on different
environmental cues are species where the effect of temperature has been shown to vary
with other environmental or maternal factors. For example, there is evidence from turtles
and lizards that the maternal phenotype influence sex determination in TSD species (e.g.,
Radder et al. 2009; Schwanz et al. 2010; Warner et al. 2007). Although there is little evidence that this is due to maternal allocation of hormones per se (Radder et al. 2009), it
could involve other gene products or environmental compounds that promote or interfere
with regulation of, for example, aromatase. This may require very limited changes at the
level of genetic networks of sex determination in the gonad itself. Indeed, endocrine disruptors show the potential for incorporating novel environmental input in sex determination in both ESD and GSD species.
FROM ESD TO GSD AND BACK AGAIN
The most conspicuous evolutionary changes in sex determination are transitions between
ESD and GSD, a transition that is probably quite common. In vertebrates, ESD evolved
independently in fish (Mank et al. 2006) and reptiles (Pokorná and Kratochvíl 2009).
However, TSD may be ancestral in extant reptiles, suggesting that there are at least six
cases of independent evolution of GSD from TSD in turtles, and probably several transitions in both directions in lizards, some of which must be very recent (Organ et al. 2009;
Pen et al. 2010; Pokorná and Kratochvíl 2009).
Despite the prevalence of ESD–GSD transitions, the origin and evolution of the regulatory processes that underlie transitions between GSD and ESD are poorly understood
both theoretically and empirically. A major problem is that most of what we know about
the molecular mechanisms of sex determination comes from studies of humans, mice, and
Drosophila, and these organisms may not be representative of clades that show evolutionary transitions between GSD and ESD (e.g., mammals are unusual vertebrates in that sex
determination apparently is not hormonally regulated and that almost all species have
heteromorphic sex chromosomes). This leaves us far more questions than answers; here
we only address two of the most fundamental ones.
First, are the networks underlying ESD and GSD similar, or are they built on entirely
different premises? Perhaps the most straightforward way to change a GSD system to an
ESD one would be to alter the promoter from constitutive to inducible so that its expression becomes environment-dependent. For example, Dmrt1 is implicated in sex determination in many vertebrates, including species with GSD and TSD. A duplicate of this gene
occupies a central node in sex determination in some fish species with an upstream mechanism involving dosage effects (Matsuda et al. 2002; Nanda et al. 2002), suggesting that
TSD could originate via mutation in this gene, making its expression temperaturedependent. If TSD is under positive selection, this may subsequently result in evolutionary
loss of genetic variation (and hence heterogamety) and evolution of a system with strong
environmental effects on sex determination.
Dmrt1 may also be involved in transitions in the opposite direction. In birds, the gene
sits on the Z chromosome and is therefore more highly expressed in males (the homogametic sex) and seems to occupy the most upstream position in the sex-determining network
(Smith et al. 2009). Consequently, this configuration could be a system derived from a
reptile TSD system with Dmrt1 being the main temperature-sensitive gene. However, it is
equally possible that the avian sex determination network has evolved from a system where
one or several other genes involved in the initiation and maintenance of testes or ovarian
From ESD to GSD and Back Again developmental pathways exhibited temperature sensitivity in their expression. Regardless,
Dmrt1 may have been a particularly likely target for mutations of major effect, given its
particular function as encoding transcription factors and its evolutionary conservation in
gonad differentiation (Graves and Peichel 2010).
Overall, it would seem that the main constraint on evolutionary shifts between GSD
and ESD is to maintain the self-sustaining expression of genes to prevent the production
of ovotestes. Consequently, we might expect that transitions from GSD to ESD could
involve complete loss of upstream master triggers as long as one or several key elements
in the downstream feedback loop show consistent expression levels under different environmental conditions. This could involve mutations directly in those genes or in genes
originally being outside of the sex determination network. For example, it is possible that
changes in rates of cell proliferation in the gonad can shift the time window of hormone
exposure to expose environmental effects on sex determination even if variation in cell
proliferation does not ancestrally appear to have a causal effect on sex determination (Uller
and Helantera 2011). A perspective that emphasizes the many different targets for mutations that can influence sex determination (Sarre et al. 2004, 2011) is consistent with the
observation that species with TSD often show substantial genetic variation in the extent
to which temperature affects gonad development (e.g., Baroiller et al. 2009; Rhen et al.
2011; Warner et al. 2008).
Alternatively, ESD may originate as epigenetic changes in genes involved in selfsustaining feedback loops, which could subsequently become stabilized and refined via
selection on novel or existing genetic variation involved in epigenetic regulation (Uller
and Helantera 2011). This also raises the possibility that GSD systems could evolve from
ESD systems via the evolution of stronger genetic effects on DNA methylation of key
genes in existing sex-determining systems. At this point, however, these scenarios remain
speculations, and we do not know if transitions between GSD and ESD involve simple
changes in master triggers, or whether the entire network, or some substantial portion of
it, is being rewired to accommodate evolutionary shifts between different systems (see
also Crews and Bull 2009; Sarre et al. 2004, 2011; Uller and Helantera 2011).
Second, are transitions between ESD and GSD as common as the reverse? Comparative data suggest that transitions from ESD to GSD are far more common than the reverse,
although this is confounded by different rates of speciation and extinction (Organ et al.
2009; Pokorná and Kratochvíl 2009). If this pattern is indeed true on a broad scale, it may
indicate that the required network changes for the different transitions are not equally easy.
Sex chromosomes have been referred to as evolutionary traps (Pokorná and Kratochvíl
2009), suggesting that transitions to GSD may be more common than transitions from it.
Aside from the problems described earlier associated with turnover of heteromorphic
chromosomes, there are few a priori reasons to assume that GSD based on homomor­
phic sex chromosomes prevents transitions back to ESD. However, our understanding of
these transitions is hampered by the fact that sex-determining mechanisms are often
unknown in the most interesting and informative clades. Advances in recording mode of
sex determination, as well collecting these records into a comprehensive phylogeny of sex
determination, would represent a major advance in understanding rates of transitions
between different systems.
Suggestions for Future Work
First and foremost, a key component for understanding the evolution of sex determination
is to actually understand how sex is determined in a broad array of animals. In this regard,
29
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The Evolution of Sex Determination in Animals
the model systems for sex determination, namely mice, humans, and Drosophila, may not
be the most informative because they are present in clades where sex determination is
highly conserved. Only when we understand the networks underlying sex determination
can we study how the networks change; therefore, it will be more informative to study
clades where sex-determining mechanisms change relatively often.
The phenotypic aspects of sex determination are also of potential interest. For example,
in some animals, somatic and gonadal sex has been, to a large extent, decoupled. Clear
examples of this exist in fish with multiple male reproductive tactics, where males may
mimic females in phenotype to dupe a resident male into giving them access to the nest
(Mank and Avise 2006) and the opportunity to steal fertilization events. How do female
mimics disassociate gonadal sex determination from somatic sexual phenotype? How have
they partitioned the genetic network of sex determination into separate gonadal and
somatic forms? In some ways, these female mimics can be thought of as intersex individuals, and this leads to questions regarding the origin of cross-sexual transfer of developmental pathways (West-Eberhard 2003), for example, how can changes in the genes
underlying maleness and femaleness arise without causing disruptions in the sex determination pathway and suboptimal male and female phenotypes?
Beyond this call for more data that will enable comparative studies of sex-determining
systems in greater detail, there is also a paucity of theoretical studies that explicitly model
the evolution of sex-determining regulatory networks (as opposed to the selective advantage of GSD versus ESD). This will not only provide more rigorous assessment of verbal
arguments, but could also generate directional predictions that can inform empirical work.
Recent explicit focus on the evolution of the regulation of sex determination (rather than
the selective context that favor genotypic versus environmental control) provide a promising start for being able to understand and predict patterns of sex determination, both in
terms of developmental mechanisms and selection (e.g., (MacCarthy et al. 2010; Pen
et al. 2010; Quinn et al. 2011; Uller and Helantera 2011).
ACKNOWLEDGMENTS
J. E. M. is grateful for support from the BBSRC and the ERC under the Framework 7
Agreement (grant agreement 260233). T. U. is grateful for funding from the European
Union’s Seventh Framework Program (FP7/2007-2011) under grant agreement 259679.
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