Temperature- Dependent Sex Determination (TSD) in Vertebrates: benefits
and disadvantages, an evolutionary prospective- Outline
Introduction:
 Explain TSD: how its tested for, which organisms exhibit it
 Independent evolutions (some squamates exhibit it, some don’t)
 Couple different organisms (viviparous lizard, fish, short-lived lizard…)
 Introduce 5 hypothesis
Discussion: [advantages/disadvantages for each hypothesis]
 Alternative explanations for adaptive significance:
o Phylogenetic inertia: TSD is an ancestral form of sex determination
with no current adaptive significance. This hypothesis is difficult to
test but seems inconsistent with the phylogenetic lability of sexdetermining systems in reptiles. (Shine 1999)
o Group-adaptation: TSD has evolved because it facilitates group
fitness through sex-ratio skewing. This hypothesis relies upon the
onerous assumptions inherent in group selection, is an argument for
sex-ratio bias rather than any specific sex-determining system and is
not consistent with available data on population structure of TSD
reptiles. (Shine 1999)
o Inbreeding avoidance: by resulting in single-sex clutches from
natural nests, TSD reduces deleterious effects of inbreeding among
siblings. This hypothesis is inconsistent with the prevalence of TSD in
long-lived iteroparous organisms (such as crocodilians and many
turtles), where offspring from many annual cohorts interbreed. (Shine
1999)
o Differential fitness: if incubation temperatures differentially affect the
fitness of male and female offspring, TSD can enhance maternal
fitness by enabling the embryo to develop as the sex best-suited to
those incubation conditions. This hypothesis is logically more robust
than the preceding ones and seems the most consistent with available
data. However, it has not been widely appreciated that the ‘differential
fitness’ models actually include a considerable diversity of separate
hypotheses that advocate quite different mechanisms as to why sex and
incubation temperatures can have an interactive effect on organismal
fitness. (Shine 1999)
 Quasi-neutral Hypothesis: TSD is maladaptive, but not strongly enough to
be selected against. (Girondot and Pieau’s, Shine)
Conclusions:
 Select theory with strongest support (maybe one stronger for short-lived
organisms, one stronger for long lived)
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Timothy K. Shrout
Juniata College
Reproductive Biology 2006
Dr. John Matter
Temperature- Dependent Sex Determination (TSD) in Reptiles: benefits and
disadvantages, an evolutionary prospective
Introduction:
“Environmental mechanisms of sex determination (ESD) are defined as those
instances where an offspring’s sex is shaped after fertilization by factors in the external
environment” (Janzen and Paukstis 1991). ESD is present in both plant and animal taxa
in some form, and many of these examples fit well with current theories. Other incidents
of ESD, however, remain ambiguous with respect to their evolution (Shine1 1999). The
main occurrence of ESD in vertebrates is that of temperature-dependent sex
determination (TSD), in which the incubation temperatures of certain species during
development influence the sex of the resultant offspring. Temperature sex determination
has been discovered in some lizards, most chelonia and all crocodilia. TSD was
historically known from long-lived reptilian species such as the testudinata and the
crocodilian, but has recently been described in several species of fish and short-lived
lizards.
It is thought that TSD is controlled through differential concentrations of sex
steroid hormones in developing embryos. At certain species-specific temperatures the
aromatase enzyme shows higher activity in the developing embryo. This up-regulation
occurs either through a temperature-dependent activity, or temperature-dependent gene
transcription. Both methods result in higher concentrations of estrogens in the embryo,
resulting in the feminization of gonadal tissues. This theory has been supported with the
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use of experimental dosing studies in many different species. When estrogen was applied
to eggs at a critical time during development, it was shown that females develop
regardless of temperature. These low levels of estrogen used in the dosings are in
proportion to the low levels which would be produced by the embryo, if its aromatase
enzyme activity had been up-regulated by the proper incubation temperature. (Janzen and
Paukstis 1991)
TSD is difficult to document and often requires research both in lab and field
settings. One laboratory method requires that a chromosome squash is performed which
should expose any sex chromosomes, if present. Secondly, if no distinguishable sex
chromosomes are present, sex ratios are compared across offspring which were incubated
in a wide array of temperatures. A significant shift in the ratios, from the expected 50:50,
which corresponds to the varied incubation temperatures would suggest that temperature
played a roll in the organism’s sexual development.
TSD has become a conundrum to evolutionary biologists and ecologists due to its
fickle nature and erratic presence in the evolutionary tree of reptiles. This erratic
distribution has led to multiple theories on the evolution of TSD, or the evolution of
genetic sex determination (GSD) as it may be, due to the fact that it is currently unknown
which mechanism arose first. Janzen and Paukstis, found that to assume ESD is the
ancestral state for chelonians, there are a minimum of 7 evolutionary events to bring
about the current distribution of GSD and TSD in the order (Janzen and Paukstis 1991).
They found that to assume GSD as the ancestral state, a minimum of 13 events must have
occurred (Janzen and Paukstis 1991). The common practice in cladistical analyses is to
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choose the tree which relies on the smallest number of assumptions, thus one might come
to the conclusion that TSD is the ancestral state for reptiles.
Many lizards and most snakes exhibit GSD, but there are many documented cases
of GSD and TSD being present in the same family, which seems to suggest the
independent evolutions of different sex determining mechanisms. It was recently shown
that “TSD has evolved independently in at least two lineages of montane lizards that are
viviparous (live bearing) rather that oviparous (egg laying)” (Langkilde and Shine 2005).
These variable appearances of TSD and possible independent evolutions suggest that the
different traits are highly variable and under strong selection pressures (Janzen and
Paukstis 1991). There are currently several theories as to the origin of these pressures
and the advantages of TSD, which attempt to explain the persistence of the two different
mechanisms.
Discussion:
Currently there are five partially accepted theories as to the evolutions of TSD in
reptiles. One of these is the phylogenetic inertia hypothesis. This hypothesis asserts that
TSD “might persist in some reptiles because they lack the genetic variation which would
allow evolution of GSD” (Ewert and Nelson 1991). TSD is explained using this
reasoning as the ancestral state, and many phyla lack the genetic variation and selection
pressures to evolve GSD. This theory remains plausible when one mechanism of sex
determination was believed to signify higher reptilian taxa, but new evidence suggesting
novel arisals of both TSD and GSD, especially in turtles and lizards, suggests that neither
form is more ancestral than the other (Ewert and Nelson 1991).
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One reason for the persistence of this theory is that in tropical environs, the
variability of nest temperatures for certain organisms could be very low, resulting in the
need for a very small variance between male and female producing temperatures. This
amount of specificity, it was hypothesized, would be difficult to produce with low levels
of genetic diversity. However it has been shown, especially in turtles, that there is a large
heritability in narrow transition zones, and when combined with transition zones differing
from one degree Celsius to greater than four degrees in some phyla. Due to this degree of
variance across populations, it must be assumed there are high levels of genetic diversity.
Thus there is strong evidence that phylogenetic inertia is not a sufficient explanation for
the distribution of TSD and GSD in many reptiles. (Ewert and Nelson 1991)
Another, related theory is that of the Quasi-neutral hypothesis. This hypothesis
and its supporters view GSD as a more advanced mechanism for sex determination, and
assert that TSD only persists because it is not selected against; however, they state that
TSD provides no advantages at the same time. Girondot and Pieau, the main supporters
of this hypothesis assert that intersex gonads can occur in animals exhibiting TSD, and
that sex ratios can fluctuate greatly over several years due to differences in climate
(Girondot and Pieau 1999). As Shine points out though, recent findings show that there
are many short-lived species which have now been shown to posses TSD. These shortlived species seem to suggest that environmental temperature fluctuations from year to
year do not alter the sex ratio enough to create a disadvantage, or else they could not
persist while relying on TSD. Shine also reiterates that “transitions between GSD and
TSD have occurred frequently in reptilian phylogeny,” which would not occur if GSD
was in actuality superior, or “better” suited than TSD. (Shine2 1999)
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The last argument that the proponents of the Quasi-neutral hypothesis propose
involves the heritability of threshold temperatures, which illustrates in a genetic influence
on the underlying mechanism of temperature-dependent sex determination. It has been
found that there are heritable differences in threshold values within species across
geographic space. Girondot and Pieau argue that this genetic component would lead to
development of embryos towards the “wrong” sex. They then go on to contend that the
presence of this heritability shows that GSD, at some level, must be a better adapted
system than TSD. Shine once again retorts by saying that “we would expect to see
transitional cases wherein sex was determined not simply by incubation temperature or
genetics, but by a combination of the two (Shine2 1999).” Thus, it becomes necessary to
acknowledge a genetic component, but not to argue that the presence of such a
component affirms the superiority of GSD over that of TSD.
Another hypothesis for the evolution or maintenance of TSD is that of groupadaptation. This hypothesis, proposed by Ewert and Nelson, argues in favor of TSD
because it would “promote group fitness through sex-ratio skewing” (Shine 1999). The
sex ratio skewing is towards a higher number of females present in a population. This
hypothesis revolves around several large assumptions. The first is that there are low
levels of gene flow in the populations, which would avoid adaptive advantage gained by
immigrant males entering a female biased group (Ewert and Nelson 1991). The second
assumption is that the groups experiencing low levels of gene flow “must be re-founded
periodically” (Ewert and Nelson 1991). This would allow the maintenance of female
biased ratios. It is thought that if these conditions are satisfied, female biased ratios
would occur to enhance group productivity upon the founding of new populations. Thus,
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selective forces would promote the creation of nests which would result in incubation
temperatures favoring female offspring. This change in behavior and skewing of sex
ratios would only be possible with TSD, because in the presence of GSD, constant ratios
(nearly 1:1) would occur by default. (Ewert and Nelson 1991)
Ewert and Nelson go on to discredit this hypothesis though, in later stating: “the
high proportion of significant, extreme, or modest male-biased values (pertaining to
studies of adult sex ratios) and the paucity of significant or extreme female-biases do not
appear to support a theory of periodic re-founding” (Ewert and Nelson 1991). According
to Shine, the group-adaptation hypothesis is weak in that it relies on too many
assumptions inherent in the ideas of group selection, and that this hypothesis is an
argument for sex ratios biases, rather than a specific sex-determining system (Shine
1999). Shine goes on to assert, much like what Ewert and Nelson said, that the
hypothesis is not supported by current data on population structures of reptiles with TSD
(Shine 1999). These reasons, along with the fact that this hypothesis may not be viable
for more than one species in a given area- for instance in the US, alligators have hot
males and cold females, while many turtles have hot females and cool males- lend
support for the presence of other mechanisms involved in the evolution of TSD.
Yet another proposed method for the evolution and maintenance of TSD in
reptiles is that of inbreeding avoidance. This theory contends that TSD is present so that
nests result in unisex clutches, thus inbreeding between full siblings would become
highly unlikely (Ewert and Nelson 1991). It is known that unisex clutches frequently
occur in natural settings, and in species that have low rates of emigration, TSD would
work against the negative effects of inbreeding by creating unisex clutches (Ewert and
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Nelson 1991). This however seems unlikely as a primary mechanism due to the fact that
many species exhibiting TSD are long lived, and unless completely random mating
occurs in a population, it is likely that close sibling mating could occur from cohorts
originating in different years, especially with low rates of emigration (Shine 1999). This
theory also breaks down among reptiles that produce more than one clutch per year.
These clutches often result in offspring of different sexes, while the female used the same
sperm from the first mating. Lastly, unisex clutches do not occur in all settings, and with
long-lived species, parent-offspring mating could also be possible. Thus, unisex clutches,
resulting in sib-avoidance are not strongly selected for, and this aspect of TSD is viewed
only as an added advantage, not a driving force.
The final and most strongly supported explanation for the evolution of TSD in
reptiles is that of differential fitness that was originally proposed by Bull et al., whereby
sex determination and some environmental factor are linked because the environmental
factor has a differential impact on the fitness of male versus female offspring (Warner
and Shine 2005). This theory purposes a link between developmental temperature and
lifetime fitness through differential phenotypic traits produced in offspring at different
temperatures. The most common factor which is suggested as the mode for this selection
is post-hatchling growth and survivorship, whereby offspring of different sexes produced
at different temperatures, and possibly from different egg sizes, show increased or
decreased levels of fitness (Warner and Shine 2005).
Warner and Shine found that differential fitness is related to nest temperatures in
the jacky dragon, and short-lived Australian species exhibiting TSD. They found that
clutches laid earlier experienced higher temperatures during incubation and hatched
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earlier, which resulted in a longer period of time in which to grow before the oncoming
winter. The jacky dragon exhibits medium male, hot and cold female pattern, meaning
that the warmer, quicker hatching eggs would be mostly female. It was found that “these
early-hatched (warm incubation) individuals were substantially larger by the onset of
winter than were individuals from the cool incubation” (Warner and Shine 2005). They
also asserted that earlier hatched individuals were smaller upon hatching and exhibited
higher mortality rates than their cool incubated counterparts. These influences were
attributed to a possible increased rate of development or an increased exposure time to
predators. In the end Warner and Shine state that “seasonal timing of hatching (related to
incubation temperature both as a direct effect, and as a correlate of high soil temperatures
in early-season nests) is likely to have a major impact on fitness” (Warner and Shine
2005).
An experiment done with Alligator mississippiensis found that male hatchlings
from male-producing temperatures grew faster than males induced from female
temperatures using aromatase inhibitors. The same was also true for females and their
respective temperatures. This study found several other interesting facts as well. The
hatchlings produced at intermediate temperatures (50:50 sex ratio in nature) grew the
fastest, which would push populations towards a 50:50 sex ratio. The one thing this
study overlooked is the possibility that the aromatase inhibitors and estrogens used to
induce hatchlings of a certain sex from the wrong temperature could have a detrimental
influence on fitness and growth rates. (Janzen and Paukstis 1991)
Langkilde and Shine studied another lizard in Australia (Eulamprus heatwolei)
that exhibits both TSD and viviparity (live birth) the presence of both the reproductive
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regimes theoretically allows the mother to knowingly influence the sex and phenotype of
her offspring through her thermoregulatory capabilities. This species has the capability
to produce litters anywhere between 0% and 100% male. It was observed that larger size
at birth, resulting from cooler, more variable maternal temperature, enhanced the survival
of male offspring, but reduced the survival of the females. It was also determined that
males and females within a litter were also influenced in the same way by maternal body
temperature. When a mother produced larger male offspring, she also produced larger
female offspring. The researchers proved that mothers could shift the sex ratio of their
offspring if their basking abilities were limited, so that their litter acquired optimal traits.
(Langkilde and Shine 2005)
Lastly, a study performed by Janzen in 1995 on sex determination and mortality
in turtles found a correlate between optimum egg temperature and survivorship. It was
determined that hatchling Chelydra serpentina produced at extreme temperatures (26 o
and 30o C) showed higher rates of survivorship due to their tendency not to run (Janzen
1995). In hatchling snapping turtles, the best anti-predator defense is to remain cryptic,
thus when individuals are more likely to attempt to flee, they are more likely to die.
These extreme temperatures happen to be the all male and all female producing
temperatures, implying that increased survivorship results from nests with 100% male or
100% female sex ratios. This would promote TSD, in that offspring produced at these
temperatures were better adapted than same sex offspring produced at intermediate
temperatures. Thus a female is able to influence the survivorship of her nest when she
chooses the nest location. (Janzen 1995)
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The selection pressures acting on the evolution and maintenance of TSD in
reptiles are still a mystery, but then again so are the selection pressures acting on the
evolution of any system or organism which is currently known. What has been
determined is that TSD was most likely the ancestral state if we use cladistics and the
evidence found in turtles and members of the crocodile family. One of the main reasons
TSD is so difficult to study is because it has appeared or been retained so sporadically
throughout taxa, that no one mechanism could be correct.
It is probably safe to assume that phylogenetic inertia, the quasi-neutral, and the
group adaptation hypotheses are the least likely mechanisms, as they have the least
experimental support throughout the various studies. In contrast sib-avoidance seems an
added benefit of TSD, probably contributing to its persistence, but most likely is not the
driving force in long-lived species. The differential fitness model has been illustrated in
natural settings on more than one occasion, and seems to be the most likely mechanism.
Even so our current understanding involving the pressures influencing the persistence of
TSD and the evolution of GSD are barely known. This is a problem inherit within the
study of evolution, in that it is impossible to observe the traits and conditions of the
systems in which these characteristics evolved. If we wish to pursue this topic further
more work must be done first on the mechanisms through which TSD works, then on the
adaptive significance these mechanisms can imbue within the offspring produced. Until
we gain a better understanding of these topics, we will be unable to determine the
possible evolutionary mechanisms.
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