APPLIED ANIMAL

BEHAVIOUR
SCIENCE
ELSEVIER Applied Animal Behaviour Science 5 1 ( 1997) 25 1-265
Environmental sex determination in reptiles
Claudio Ciofi Ian R. Swingland
a Institute of Zool ogj . Zix~logicul Society oj l.ondon. Regent'.\ Park. London. NWI 4RY. UK
The Durrell Institute ojConservation and Eciilofl. University (i fKent . Canterbury. C72 7PD. UK
Abstract
In recent years, representatives of more than 40 families of reptiles have been studied to
understand how environmental parameters affect sex determination. In this review, we summarise
the distribution and taxonomic pattern of sex determining mechanisms, outline the main hypothe-
ses of the adaptive significance of temperature sex determination (TSD), and of skewed population
sex ratios. We also examine the competing hypotheses of the physiological and molecular
mechanisms involved in TSD. 0 1997 Elsevier Science B.V.
Ke p v r d . ~ : Environmental sex determination; Genotypic sex determination; Temperature; Reptiles
1. Introduction
A remarkable diversity of sex determination systems is found among different animal
taxa. In most cases, sex is determined chromosomally. The mechanisms involved have
been under dispute since the first genetic system of sex determination was described in
the fruit-fly Drosophila melanogaster (see Wachtel, 1993). In mammals. this debate has
been resolved with the recent discovery of the SRY gene on the Y chromosome as the
testis-determining factor (see Hawkins, 1993 for a review).
While genotypic sex determination (GSD) depends on genetic factors alone, environ-
mental sex determination (ESD) depends on post-fertilisation environmental factors. In
recent years, the sex of an increasing number of animals has been found to be influenced
primarily by the environment, and ESD mechanisms have been examined in a variety of
taxa (rotifers. nematodes, polychaetes and echiurids: Korpelainen. 1990; crustaceans:
Rigaud et al., 1991; fishes: Conover et a]., 1992; Beamish, 1993).
Corresponding author. Conservation Genetics. Institute of Zoology, Zoological Society of London,
Regent's Park, London NW1 4RY, UK. Tel.: +44 171 449 6618; fax: + 4 4 171 586 2870; e-mail:
c.ciofi@ucl.ac.uk.
0168-1591 /97/S17.00 Published by Elsevier Science B. V.
Pl l S0168- 1591( 96) 01 108- 2
25 2 C. Ciofi. I. R. S~inyliintI/AppliedAnimal Behaviour Science 51 11997) 251-265
Reptiles exhibit different modes of both GSD and ESD. sometimes occurring together
even within a single family (Ewert et a]., 1990: Janzen and Paukstis. 199la). Modes of
GSD include at least three patterns of heterogamety in both sexes; XX/XY, XXX/XXY,
and pseudo XO are forms of male heteroganiety. while ZZ/ZW, ZZZ/ZZW, and
ZZZ/ZWW are types of female heterogamety. In addition. morphologically undifferen-
tiated sex chromosomes have been found to occur in several families (Olmo. 1986).
Among environmental parameters, egg incubation temperature appears to be the most
critical factor affecting gonadal differentiation in reptiles. Temperature sex determina-
tion (TSD) has been recorded in chelonians (turtles and tortoises), lizards, crocodilians
(alligators and crocodiles) and, recently, tuataras. All species of snakes and amphisbae-
nians examined to date have been shown to have only GSD mechanisms (Janzen and
Paukstis, 199 la).
Since Chamier (1966) first described the effect of temperature on sex ratio of the
hatchlings in the lizard, Agama agama, there has been a series of papers describing the
importance of temperature on the sex ratio of lizard and chelonian embryos (e.g. Pieau,
1971, 1972, 1974; Ynetema, 1976). Subsequently, several studies have focused on the
ecological and evolutionary implications of TSD in reptiles and its effect on sex ratios in
natural populations (e.g. Nichols and Chabreck. 1980: Ewert and Nelson, 1991; Burke,
1993), while an increasing amount of research has attempted to uncover the molecular
and physiological mechanisms underlying TSD (Deeming and Ferguson, 1989;
Etcheberger et a]., 1991 ; Desvages et al.. 1993; Crews et al., 1994; Pieau et al., 1994a;
Pieau, 1996). Indeed, some of these studies have been carried out in zoological gardens
(e.g. Hammack, 1989; Horn and Visser. 1989; Boyer et al., 1989; Hairston and
Burchfield, 1992), where sex ratio manipulation by controlling incubation temperature is
crucial in captive breeding programmes of threatened or endangered reptiles.
Potentially, zoos represent an important platform by which TSD theory and experi-
mental designs on TSD can be tested in a practical setting. This review outlines the
pattern of distribution of TSD in reptiles. its adaptive value and the relationships of sex
determining mechanisms to the ecology of this taxa. Experiments investigating how
temperature affects sexual differentiation are also described.
1.1. Distribution and taxonomic patterns of sex determining mechanisms
More than 70 species belonging to 43 families of reptiles have shown TSD either in
laboratory experiments or in field studies. Chelonians and lizards exhibit large variation
in sex determining mechanisms (Fig. 1). In chelonians. although Chelidae, Trionychidae
and Staurotypidae show GSD only, TSD is prevalent in most other families studied.
Within the Emididae, both Batagurinae and Emidinae show both GSD and TSD, though
the latter is much more common (Janzen and Paukstis. 199la). However, for some
species, the sex determining mechanism is still uncertain. One species of the Chelidri-
dae. the alligator snapping turtle Macroclenzys renznzinckii, has been reported to show
TSD (Packard and Packard, 1988). although Ewert et al. (19941, report that females are
produced even when the incubation temperature is shifted among male-producing values
during development. Honiomorphic sex chromosomes seem to be a necessary pre-
requisite for TSD (Bull, 1980), while GSD may occur under homo- or heteromorphy
(Palmerallen et al.. 199 1).
C. C~o f i . I.R. Swi ndunf i / Appl i ed Animal Behuuiour Science 51 (1997) 251-265
ESD GSD Genotypic
mechanism
Pelomedusidae
Chelidae . Htg, Hmg
CHELONIA
I
Cheloniidae
Dermochelyidae
Trionychidae
Carenochelyidae
Staurotypidae
Dermatemydidae
Kinosternidae
Emydidae . . Hlg, rtmg
Testudin~dae
1 CROCODYLIA
Agamidae . .
lguanidae . Htg Hmg
Chamaeleontidae .
Eublepharidae . .
Pygopodidae Htg
SAURIA
Gekkomdae . . Hlg. Hmg
1 1 _____ AMPHISBAENIA "tg
L-l -
SERPENTES Htg, Hmg
Teiidae Htg, Hmg
Lacenidae . . Htg, Hmg
Anguidae
Varanidae . . Hlg
Fig. I . Phylogenetic tree for reptiles showing those taxa studied for sex-determining mechanisms. ESD,
environmental sex determination; GSD, genotypic sex determination. Genotypic mechanisms refer to the
presence of heterogametic (Htg) or homogametic (Hmg) sex chromosomes.
In lizards, GSD occurs in both iguanids and scincids, where male heterogamety
appears to be ubiquitous. Both GSD and TSD have been described in the Agamidae,
Eublepharidae. Gekkonidae, and Lacertidae, while the coexistence of the two mecha-
nisms is still uncertain for Iguanidae and Varanidae (Viets et al., 1994). The recurrence
of GSD in these taxa may be partly due to the presence of viviparous and partheno-
genetic species, whose reproductive mechanisms presumably require genetic input for
sex determination (Bull, 1980). In Chamaeleontidae, Chamaleo pardalis probably has
GSD, while Rhampholeon k. kerstenii may have both GSD and TSD (Viets et al., 1994).
All 11 species of crocodilians examined to date show TSD (Deeming and Ferguson,
254 C. Cioft. I.R. Sw~ngl und/ Appl r ed Ani muf Behuvmur Science 51 119971 251-265
a) Lizards
b) Chelonians Crocodilians ( 7)
- -- --
20 40
Temperature ( C)
Fig 2 Hatchling sex ratio as a function of incubation temperature in reptiles
1989; Lang and Andrews, 1994). Among tuataras, TSD has been recently reported for
Sphenodon punctatus (Cree et al.. 1995). Only one species of amphisbaenians has
heteromorphic sex chromosomes (female heterogamety), and none has been investigated
for ESD. Three species of snakes have been examined to date. They have either female
heterogamety or homomorphic sex chromosomes; none has been shown to have TSD
(Nerodia fasciata, Osgood, 1980; Dunlap and Lang, 1990; Lampropeltis triangulum
andesiana, Hammack, 1989; Pituophis melanoleucus, Burger and Zappalorti, 1988).
Three different patterns of TSD have been described in chelonians, crocodilians, and
lizards. thus far.
Eggs incubated at constant, low temperatures produce only females, whereas those
maintained at constant, higher temperatures produce only males. Intermediate temper-
atures produce both sexes. This pattern is found in most species of lizards (Fig. 2(a)),
and has been reported in crocodilians by Ferguson and Joanen. 1982; Ferguson and
Joanen, 1983; but see pattern 3 below). They describe three different types of nest
site in the natural habitat of Alligator mississippiensis: wet marsh, dry marsh, and
elevated firm ground. The latter are hot (34'C) and produce approximately 100%
males. Wet marsh nests are cool (30C and produce approximately 100% females.
Dry marsh nests have intermediate temperatures, producing males in hot sites, and
females in cold ones.
Females are produced at high incubation temperatures and males at low temperatures
in many chelonians (Fig. 2(b)). In Emys orbicularis all the animals are male below
27.5OC. whereas 100% are female above 29.SC Between 28OC and 29OC, both
sexes and some intersexes appear (Pieau and Dorizzi, 1981; Pieau, 1982).
Females are produced at low and high temperatures. with males produced at
intermediate temperatures. This occurs in some species of chelonians, lizards, and
many species of crocodilians (Fig. 2(c)). In the leopard gecko Eublepharis macular-
C. Cioji. 1.R. S~l und/ Appl i e dAni nui l Behuuiour Science 51 11997) 251-265 255
ius, females are produced when the eggs are incubated at 26 O C (loo%), 30 (70701,
and 34OC (95%), whereas males (75%) are predominantly produced between 30 and
34OC (Deeming and Ferguson, 1988; Ewert and Nelson, 1991; Flores et al., 1994). In
the snapping turtle Chelydra serpentina, incubation at 20 and 30 results in 100%
production of females, whereas a majority of males are produced between 22 and
28OC (e.g. Pieau, 1976; Bull, 1980). Additionally, Lang and Andrews (1994) report a
similar pattern in crocodilians (Alligator mississippiensis. Caiman crocodilus, and
seven species of the genus Crocodylus). For example, they incubated fertile eggs of
Alligator mississippiensis at constant incubation temperatures, resulting in 100%
production of females below 31 OC and above 34OC, while 100% males were
produced at 33 OC (but see Deeming and Ferguson, 1989, 1991).
As we can see from these examples, selection for mode of sex determination varies
extensively among reptilian groups. However. as GSD and TSD co-occur in some taxa,
both modes seem to be viable evolutionary strategies. An interesting array of studies has
been carried out concerning the origins of these two mechanisms, and has been reviewed
by Janzen and Paukstis (199la). TSD and GSD seem to occur with the same frequency
among reptilians, and both are present in living members of families represented among
the earliest known fossil chelonians. Thus far, it has been difficult to determine which
character state (TSD or GSD) is ancestral, and there is the possibility of either multiple
or independent origins of TSD and GSD within reptilians.
1.2. Adaptive significance of ESD
The prerequisite for the persistence of ESD in reptilians seems to be related to a
thermally patchy environment, in which different incubation temperatures in different
microhabitats allow natural populations to produce hatchlings of both sexes with
potentially different physiological characteristics (e.g. different metabolic rates or poten-
tial growth rates).
A patchy distribution of the environmental factors (e.g. temperature) involved in
embryo sex determination can also affect the lifetime fitness of hatchlings in different
ways. To explain the adaptive significance of ESD, Chamov and Bull (1977) proposed a
model where hatchling fitness varies most among patches and between sexes, so that
some patches provide optimum conditions for one sex but not the other. Following the
Chamov-Bull model, Webb et al. (1987) proposed post-hatching growth rate and body
size as the most important fitness traits correlating with incubation temperature and
offspring sex (see also Dunham et al., 1988; Wilbur and Morin, 1988). According to this
sexual dimorphism hypothesis, if the benefits of adult body size differ between sexes,
the sex that benefits most from being large should be produced at the incubation
temperature yielding large adults. As an example, in Alligator mississippiensis, larger
males are produced at higher temperatures, and a selective advantage has been reported
for those individuals; they control larger harems of females and produce more spermato-
zoa for a longer time than small males (Allstead and Lang, 1955; Deeming and
Ferguson. 1989; Joanen and McNease, 1989). In this hypothesis, TAD would be
favoured over GSD, as the latter would not guarantee that the eggs containing genotypi-
cally male embryos would be in optimum incubation conditions and therefore grow
256 C. Ciofi. I. R. Swin~ant!/AppliedAnimal Behaviour Science 51 (19971 251-265
faster and into larger males. TAD, on the other hand, allows the association of sex with
potential post-hatching growth rate, as both are determined by incubation conditions.
This relationship between incubation temperature and adult sexual dimorphism
suggests a temperature-dependent differential fitness explanation for the adaptive signifi-
cance of TSD. However, although this hypothesis could be regarded as a possible
explanation for some taxa, such as crocodilians, it does not hold for the chelonians and
lizards that have been examined (see below), and it fails to explain why some reptiles
have sexual dimorphism and lack TSD (Ewert and Nelson, 1991).
In chelonians, incubation conditions can affect hatchling survival and growth (Janzen,
1993). Bobyn and Brooks (1994) found that low temperatures reduce hatching success,
post-hatching survival and hatchlings' growth in the snapping turtle Chelidra serpentina.
They suggest that incubation conditions may be the most important factor affecting the
northern distribution of this species in Canada. However, no sex-linked selective
advantage for growth rate or body size was recorded. In several species of chelonians,
hatchlings do not show significant physical or developmental differences (e.g. Ewert and
Nelson, 1991), and the relationships between sex-determining mechanisms and sexual
dimorphism in adult body size seem to be inconsistent with the predictions of the sexual
dimorphism hypothesis (Janzen and Paukstis, 1991 b).
In lizards, a pattern similar to that of Alligator mississippiensis was found in the
leopard gecko Eublepharis macularius. In this species, 95- 100% females are produced
at 26 and 34OC, while males (the larger sex) are predominantly produced at 32.5'C.
Tousignant and Crews (1 995) found that male-biased incubation temperatures produced
larger female hatchlings than did female-biased incubation temperatures. In addition,
females from higher incubation temperatures were found to produce larger offspring.
Theoretically, this may result in better survival in natural situations. However, the
authors found no evidence of incubation temperature directly affecting reproductive
success.
Thus, if TSD is adaptive in these taxa, an explanation for its occurrence has to be
found in alternative traits. For example, Gutzke and Crews (1988) and Flores et al.
(1994) suggested behaviour and sexual receptivity as alternative parameters, producing
strong evidence for the adaptive significance of TSD in the leopard gecko. Male
Eublepharis macularius showed aggression in response to other individuals of the same
sex, and females from a male-biased incubation temperature were found to be more
aggressive towards males than embryos incubated at female-biased incubation tempera-
tures. Moreover, females produced at low incubation temperatures were sexually
receptive, while most females produced at warmer temperatures appeared to be function-
ally sterile.
Post-hatching physical performance has also been proposed as a fitness trait corre-
lated with sex and incubation temperature. In the snapping turtle C. serpentina (where
males and females are produced at low and high incubation temperatures, respectively),
females from higher incubation temperatures and males from lower ones both had higher
first-year survivorship than individuals incubated at intermediate temperatures (Janzen,
1995). A positive correlation was then found between hatchling survivorship and
post-hatching 'propensity to run', considered as a temperature-dependent antipredator
behaviour.
Thus, it seems that specific combinations of incubation temperature. gender and
behavioural characteristics affecting fitness do provide contributions towards a func-
tional explanation for the evolutionary significance of TSD.
1.3. Adaptive significance of biased sex ratios
Heavily biased sex ratios have been described in crocodilians (e.g. Ferguson and
Joanen, 1982, 1983; Woodward and Murray, 1993), chelonians (e.g. Servan et al., 1989;
Gibbons, 1990; Ewert and Nelson, 199 I), and lizards (e.g. Auffenberg, I98 1 ). Several
different hypotheses have been proposed to explain these patterns in natural populations
of reptiles (e.g. Ewert and Nelson, 1991). yet none to date provide a comprehensive
explanation.
Ewert and Nelson ( 1 991) reviewed field studies reporting female-biased hatchling sex
ratios in natural populations of TSD reptiles. In their 'group-structured adaptation'
hypothesis, they suggest that the observed pattern can only persist in small populations
with minimal gene flow and periodic re-founding events. This is because if the
population increases in size, the optimal sex ratio shifts toward 1: 1 (Fisher, 1930). A
female- or male-biased breeding system must be isolated to persist. because immigrants
may have higher reproductive success than residents. Nevo et al. (1984) found further
support for the group structure hypothesis in a number of taxa. Under these assumptions
of isolation, inbreeding should be common and the average level of heterozygosity low,
especially in comparison with GSD species. However, Burke (1993) found little
evidence in chelonians of a correlation between a skewed offspring sex ratio and low
heterozygosity, suggesting that there is no genetic evidence that inbreeding is more
common in TSD species. Moreover. Smith and Scribner (1990) reported a high
migration rate among breeding populations of Trachenzys scripta, suggesting that
isolated breeding units could hardly persist in this TSD species.
According to the 'sib-avoidance' hypothesis, females of TSD species produce
unisexual clutches (e.g. Lang et al.. 1989) in order to preclude sib-mating, and thus
reduce inbreeding. In this model, TSD would be particularly advantageous in species
with extremely low levels of dispersal. However. in chelonian species which do produce
unisexual clutches, dispersal is common. and females are likely to produce clutches of
different sexes when they lay eggs in nearby nests. Moreover, as female chelonians can
store sperm for more than 1 year, eggs laid in sequential clutches may be full sibs.
Moreover, Burke (1993) reported similar average heterozygosity values between TSD
and GSD in 20 species of chelonians. He further showed that TSD species are not more
likely to have unisex clutches.
The above hypotheses, based on eco-ethological factors associated with climatic
variation, do not seem to match the observed pattern in natural populations very well.
Servan et al. (1989) proposed the influence of genotypic mechanisms to explain the
female bias observed in Emys orbicularis. Cold incubation temperatures produce males
(see Fig. 2(b)); however, the authors found a surprising female bias in populations
sampled from cold areas. In Enzys orbicularis a male ZZ/female ZW genotypic sex
determination pattern has been proposed (Zaborski et al.. 1982. 1988). An increase of
female progeny could be explained if the sex of a few genotypic females was inverted
258 C. Ciofi. I.R. S~vi n~l und/ Appl i ed Animal Behaviour Science 51 (1997) 251-265
under the influence of temperature; that is, if a ZW (genotypic female) embryo was
exposed to low incubation temperature during the thermosensitive period (see below), it
would become a phenotypic male. When these ZW 'neo-males' mate with standard ZW
females, a majority of genotypic females (ZW, WW) will result. Indeed, Servan et al.
(1989) found that most individuals in natural populations of Emys orbicularis have
sexual phenotypes matching their sexual genotypes, but that a low percentage of
individuals are sexually inverted.
Thus, unlike the group-structured adaptation and the sib-avoidance hypothesis, the
influence of temperature on genotypic sex determination, resulting in populations with a
few sexually inverted individuals, seems to provide a good explanation for the observed
female-biased sex ratios, and could form the basis of an alternative hypothesis (see also
Bull and Charnov, 1989).
1.4. TSD mechanisms
While the evolutionary significance of TSD remains to be clarified, physiological and
molecular mechanisms are better understood. In the following sections we describe how
a temperature sensitive period during embryogenesis has been defined, and what the
main hypotheses regarding molecular mechanisms involved in TSD are.
2. Temperature sensitive stages
In several species of reptiles, temperature affects sex determination during particular
stages in embryonic development when differentiation of the gonads occurs. Preliminary
temperature-shift experiments, designed to assess the temperature sensitive period (TSP)
of gonadal differentiation during incubation, were performed by Pieau and Dorizzi
(1981) in Emys orbicularis (Fig. 2(b)). Eggs which had been first incubated at 24-26'C
for either 27, 30 or 35 days (corresponding to different stages of testicular differentia-
tion) were changed to an incubation temperature of 30C All three shifts resulted in
both males and females, with some abnormalities, showing that in 27-day-old embryos,
testicular differentiation had already begun, whereas in 35-day-old embryos, ovarian
differentiation was still possible.
To investigate further the action of temperature on sexual differentiation in embryos,
Pieau and Dorizzi ( 1981) examined the TSP for sexual differentiation by shifting the
temperature between 25 OC ( 100% male-producing temperature) and 30 OC ( 100% fe-
male-producing temperature) at different stages of embryonic development. The TSP for
both male and female differentiation was found to begin at a determinate stage in Emys
orbicularis. defined as stage 16, in which gonads are still histologically undifferentiated.
Eggs incubated at 25 OC from stage 16 to 21 produce 100% males, while 100% females
are obtained following a 30 exposure from stage 16 to 22. Shifts occurring at
different points during the TSP lead to varied proportions of males and females. In
addition, during these experiments, the incubation temperature shifts applied before and
after the TSP did not influence the sexual differentiation of the gonads.
Wibbels et al. (1991) reported a similar pattern in the alligator Alligator mississippi-
ensis, where the sensitive period encompasses 23% of the total embryonic development
C. CwJi. 1.R. Sni n~l und/ Appl i ed Animul Behuviour Science 51 < 19971 251 -265 259
period (Deeming and Ferguson. 1989; Lang and Andrews, 1994). In chelonians, this
period represents 15-25% of development, while comparable data for the leopard gecko
Eublepharis n~acularius indicate that the TSP extends for about 30% of the incubation
period (Bull, 1987).
Thus, sex ratios seem to depend on the length of time eggs are exposed to a certain
temperature regime. Moreover, temperature-shift experiments show that the duration of
exposure at the temperature required for typical male or female differentiation depends
on the stage within the TSP at which the temperature shift is initiated. The later the shift,
the longer the exposure must be. This increase in time may reflect some modification of
the gonads (male or female differentiation) due to the temperature applied between the
beginning of the TSP and the shift.
3. Molecular mechanisms
Several different theories have been proposed to explain the molecular mechanisms
behind TSD (Spotila et al., 1994). Studies highlighting the correlation between tempera-
ture and the expression of the heterogametic sex-linked H-Y histocompatibility antigen
(e.g. Zaborski et al., 1982, 1988) gave quite contradictory results (Engel et al., 19811,
and this hypothesis was eventually rejected (McLaren, 1988; Mardon et al., 1989).
Similarly, theories underlining the effect of incubation temperature on hypothalamic
control of secretion of luteinising hormone (Deeming and Ferguson, 1989), and on the
involvement of the genes coding for zinc finger proteins in the sex determination
process (Bull et al., 1988; Valleley et al., 1992) failed to provide a definitive explanation
for the genetic basis of gonadal sex determination in reptiles (Wibbels et al., 1994).
After the demonstration by Pieau (1974) of the feminising effect of the administration
of oestradiol to turtle eggs incubating at a male-producing temperature, a fourth theory,
in which gonadal differentiation is determined by the ratio of androgenic to oestrogenic
steroids, was suggested (Raynaud and Pieau, 1985; Bogart. 19871, and subsequently
corroborated (Bull et al., 1988; Dorizzi et al., 1991).
This hypothesis contradicts the organisation/default system proposed by Jost (19611,
where gender is determined by mechanisms operating on a pre-established condition. In
fact, an important concept that can be inferred from the new model is that female and
male sex determination are separate processes that are differentially affected by incuba-
tion temperatures (see below).
Several studies have produced supporting data to the steroid hormone-mediation
theory and suggested possible pathways (see Raynaud and Pieau, 1985; Crews et al.,
1989; Crews et al., 1994; Crews, 1994; Pieau et al., 1994a; Wibbels et al., 1994). The
production of the androgenic steroid dihydro-testosterone (DHT) and oestradiol is
regulated by two steroidogenic enzymes: reductase and aromatase, respectively; these
enzymes might play a key role in sex determination as they are influenced in a different
way by the egg incubation temperature (Pieau, 1974; Crews and Bergeron, 1994;
Thomas et al., 1992). For example, high levels of aromatase were recorded by Desvages
and Pieau (1992) in embryos incubated at female-producing temperatures, but not at
male-producing incubation temperatures. Moreover, the administration of aromatase
260 C. Ci oj i , I.R. Swi ngl und/ Appl i ed Animal Behaviour Science 51 119971 251-265
inhibitors produced males at intermediate and all female-producing incubation tempera-
tures in turtles (Crews and Bergeron, 1994; Wibbels and Crews, 1994) and alligators
(Lance and Bogart, 1994). The opposite pattern seems to characterise the effect of
reductase on hatchling sex ratio at intermediate male-biased incubation temperature,
where administration of reductase inhibitor produced a significant number of female
hatchlings (Crews and Bergeron, 1994).
In addition, temperature affected aromatase activity during, but not after, the ther-
mosensitive period of Emys orbicularis and Dermochelis coriacea (Desvages et al.,
19931, indicating the importance of oestrogens in gonadal differentiation in TSD
reptiles.
A key role in the activity of these steroidogenic enzymes is played by testosterone,
which has been proposed as the precursor of oestradiol by aromatase and DHT by
reductase. The fact that female hatchlings were produced when testosterone was
administered to eggs incubated at male-biased or all male-producing incubation tempera-
tures (Gutzke and Bull, 1986; Crews et al., 1989; Wibbels and Crews, 1992) suggested
the conversion of testosterone to oestradiol through aromatase. In addition, this result
was not observed using the non-aromatisable androgen DHT (Wibbels and Crews,
1992). Further evidence for this process is that the combined administration of testos-
terone and reductase inhibitor to eggs incubating at a male-producing temperature also
resulted in the production of female hatchlings (Crews and Bergeron, 1994). Similar
experiments using aromatase inhibitors (e.g. Wibbels and Crews, 1994) advocated the
importance of both enzymes in the sexual differentiation pathway. In addition, it seems
likely that temperature does not affect the aromatase enzyme activity, but directly or
indirectly controls its synthesis (Desvages and Pieau. 1992; Smith et al., 1995).
Among the models proposed to account for the role of temperature in sex determina-
tion of reptiles, the most promising is based on the action of temperature on specific
promoters associated with one or more genes encoding for steroidogenic enzymes
(Crews and Bergeron, 1994; Pieau et a].. 1994b). According to this hypothesis, at a
female-producing temperature the genes encoding for aromatase would be activated and
the genes encoding for reductase suppressed or simply remain at constitutive levels (see
Pieau, 1996) resulting in increased oestradiol production. As further evidence for this
model, Pieau et al. (1994b) recorded a positive feedback relationship between oestradiol
secretion and aromatase production at a female-producing temperature. Even if no
similar relationship has yet been found between DHT and reductase, a reverse process
should lead to activation of reductase genes and inhibition of aromatase ones at
male-producing incubation temperatures.
After the conversion of testosterone into either DHT or oestradiol, each hormone
would bind to specific nuclear receptor proteins. Each of these complexes would in turn
find a sex-specific site on the DNA, thus activating the transcription of genes associated
with the sex-determining cascade of one sex, and repressing the genes responsible for
the sex-determining cascade of the other.
Attempts to identify the genes involved in the sex-determining cascade are currently
focusing on portions of the DNA similar to the sex-specific SPY genes of mammalian
vertebrates. Tiersch et al. (1991) designed a molecular probe for the conserved region of
the SRY gene, which gave a positive signal in the DNA of reptiles with either TSD or
C. CK$, I . R. S wi n ~ d / Ap p l i e d An i ma l Behuviour Science 51 119971 251-265 26 1
GSD; however. no sex-specific pattern was revealed. A sex specific role for SRY-type
protein in two species of reptiles with TSD has been investigated (Johnson et al., 1995),
and the levels of similarity with those of mammals are being assessed (Coriat et a].,
1993). Whether these proteins have similar roles in reptiles and mammals, and where
this SRY-type gene may act in the sex-determining pathway, remain open to specula-
tion.
In conclusion, a clear understanding of exactly how temperature determines the sex
of reptiles remains elusive. Recent studies have produced data on an increasing number
of species in chelonians and crocodilians, pointing out the remarkable level of variation
in TSD patterns shown in these taxa. Much smaller proportions of lizard and snake
species have been examined. Nonetheless, the evident evolutionary plasticity suggests
that more variation in patterns of TSD, and possibly in sex-determining modes, will be
discovered as the number of species studied increases.
Acknowledgements
This work has been carried out thanks to a joint grant provided by the University of
Florence, D.I.C.E. and the Institute of Zoology. The authors are most grateful to Kate
Clark and Dr Mike Bruford for their valuable comments and suggestions during the
revision of the manuscript.
References
Allstead, J . and Lang, J.W.. 1995. Incubation temperature affects body size and energy reserves of hatchling
american alligators ( Allifutor nti.'i'.is'.ippien.\~.s). Physiol. Zool., 68: 76-97.
Auffenberg, W., 198 1. The behavioral ecology of the Komodo dragon. University Press of Florida, Gainesville.
Beamish, F.W., 1993. Environmental sex determination i n southern brook lamprey. lchthomywn m e ; . Can.
J. Fish. Aquat. Sci., 50: 1299- 1307,
Bobyn. M.L. and Brooks, R.J.. 1994. Incubation conditions as potential factors limiting the northern
distribution of snapping turtles, Chelydru serpenrmu. Can. J. Zool., 72: 28-37.
Bogan, M. H. , 1987. Sex determination: a hypothesis based on steroid ratios. J. Theor. Biol., 128: 349-357.
Boyer, D.M., Mitchell, L.A. and Murphy, J.B., 1989. Reproduction and husbandry of the bushmaster Lucht",;.~
m. mutu. Int. Zoo Yearb., 28: 190-194.
Bull, J.J., 1980. Sex determination in reptiles. Q. Rev. Biol., 55: 3-21.
Bull. J.J., 1987. Temperature-sensitive periods of sex determination in lizard: similarities with turtles and
crocodilians. J. Exp. Zool., 241: 143- 148.
Bull. J.J. and Charnov, E.L., 1989. Enigmatic reptilian sex ratios. Evolution, 43: 1561 - 1566.
Bull, J.J., Hillis, M.M. and O'Steen, S., 1988. Mammalian ZFY sequences exist in reptiles regardless of
sex-determining mechanism. Science, 242: 567-569.
Burger. J. and Zappalorti, R.T., 1988. Effects of incubation temperature on sex ratios in pine snakes:
differential vulnerability of males and females. Am. Nat.. 132: 492-505.
Burke, R.L., 1993. Adaptive value of sex determination mode and hatchling sex ratio bias in reptiles. Copeia,
1993: 854-859.
Charnier, M., 1966. Action de la temperature sur la sex-ratio chez I'embryon d' Agumu ugumu fAgumidue,
Lacertilied). C.R. Soc. Biol. Paris. 160: 620-622.
Chamov, E.L. and Bull, J. , 1977. When is sex environmentally determined'? Nature, 266: 828-830.
Conover. D.O., Vanvoorhees. D.A. and Ehtisham, A,, 1992. Sex ratio selection and evolution of environmen-
tal sex determination in laboratory populations of Mediniu mrdiniu. Evolution, 46: 1722- 1730.
262 C. CioJi. I.R. Swinglunil/Applied Animul Behuviour Science 51 (1997) 251 -265
Coriat, A.M.. Muller, U., Harry, J.L., Uwanogho, D. and Sharpe, P.T.. 1993. PCR amplification of
SRY-related gene sequences reveals evolutionary conservation of the SRY-box motif. PCR Meth. Appl., 2:
2 18-222.
Cree, A., Thompson, M.B. and Daugherty, C.H., 1995. Tuatara sex determination. Nature, 375: 543,
Crews, D., 1994. Temperature, steroids, and sex determination. J. Endocrinol., 142: 1-8.
Crews, D. and Bergeron, J.M., 1994. Role of reductase and aromatase in sex determination in the red-eared
slider (Truchemys scriptu), a turtle with temperature-dependent sex determination. J. Endocrinol., 143:
279-289.
Crews, D.. Wibbels, T. and Gutzke, W.H.N., 1989. Action of sex steroid hormones on temperature-induced
sex determination in the snapping turtle (Chelydru .lerpeiitinu). Gen. Comp. Endocrinol., 76: 159- 166.
Crews. D., Bergeron, J.M., Bull, J.J., Flores, D., Tousignant, A,. Skipper, J.K. and Wibbels, T.. 1994.
Temperature-dependent sex determination in reptiles: proximate mechanisms, ultimate outcomes, and
practical applications. Dev. Genet., 15: 297-3 12.
Deeming, D.C. and Ferguson. M.W.J., 1988. Environmental regulation of sex determination in reptiles. Philos.
Trans. R. Soc, London. Ser. B. 322: 19-39.
Deeming, D.C. and Ferguson. M.W.J., 1989. The mechanism of temperature dependent sex determination in
crocodilians: a hypothesis. Am. Zool., 29: 973-985.
Deeming, D.C. and Ferguson, M.W.J., 1991. Physiological effects of incubation temperature on embryonic
development in reptiles and birds. In: D.C. Deeming and M.W.J. Ferguson (Editors), Egg Incubation.
Cambridge University Press, Cambridge, pp. 147- 17 1.
Desvages, G. and Pieau, C.. 1992. Aromatase activity in gonads of turtle embryos as a function of the
incubation temperature of the egg. J. Steroid Biochem. Mol. Biol., 41: 851-853.
Desvages, G., Girondot, M. and Pieau, C.. 1993. Sensitive stages for the effects of temperature on gonadal
aromatase activity in embryos of the marine turtle Dermochelys coriuceu. Gen. Comp. Endocrinol.. 92:
54-61.
Dorizzi, M., Mignot, T.M., Guichard, A., Desvages. G. and Pieau. C., 1991. Involvement of oestrogens in
sexual differentiation of gonads as a function of temperature in turtles. Differentiation, 47: 9- 17.
Dunham, A.E., Miles, D.B. and Reznick, D.N., 1988. Life history patterns in squamate reptiles. In: C. GAns
and R.B. Huey (Editors), Biology of the Reptilia, Vol. 16. Alan R. Liss, New York, pp. 441-522.
Dunlap, K.D. and Lang, J.W., 1990. Offspring sex ratio varies with maternal size in the common garter snake.
Thamnophis sir~ulis. Copeia, 1990: 658-570.
Engel, W., Klemme, B. and Schmid, M.. 1981. H-Y antigen and sex determination in turtles. Differentiation,
20: 152- 156.
Etcheberger, C.R., Phillips, J.B., Ewert, M.A., Nelson, C.E. and Prange, H.D.. 1991. Effects of oxygen
concentration and clutch on sex determination and physiology in red-eared slider turtles (Truchemys
scripla). 1. Exp. Zool., 258: 394-403.
Ewert, M.A. and Nelson, C.E., 1991. Sex determination in turtles: diverse patterns and some possible adaptive
values. Copeia, 1991: 50-69.
Ewert, M.A., Etchberger, C.R. and Nelson, C.E., 1990. An apparent cooccurrence of genetic and environmen-
tal sex determination in a turtle. Am. Zool., 30: 56,
Ewert, M.A., Jackson, D.R. and Nelson, C.E.. 1994. Patterns of temperature-dependent sex determination in
turtles. J. Exp. Zool., 270: 3- 15.
Ferguson, M.W.J. and Joanen, T., 1982. Temperature of egg incubation determines sex in Alligutor
mississippiensii. Nature, 296: 850-853.
Ferguson, M.W.J. and Joanen. T., 1983. Temperature-dependent sex determination in Alliguior mi.\.si.~.sippim-
.n'.s. J. Zool. London, 200: 143- 1 77.
Fisher, R.A., 1930. The Genetical Theory of Natural Selection. Oxford University Press, Oxford.
Flores, D., Tousignant, A. and Crews, D., 1994. Incubation temperature affects the behavior of adult leopard
geckos Euh1ephuri.s muculuriu.\. Physiol. Behav., 55: 1067- 1072.
Gibbons, J.W., 1990. Sex ratios and their significance among turtle populations. In: J.W. Gibbons (Editor),
Life History and Ecology of the Slider Turtle. Smithsonian Institution Press, Washington DC, pp.
171-182.
Gutzke, W.H.N. and Bull. J.J.. 1986. Steroid hormones sex reverse turtles. Gen. Comp. Endocrinol., 64:
368-372.
C. Cioji. I.R. S\cinglund/Applied Animal Behuviour Science 51 U9971231-265 263
Gutzke, W.H.N. and Crews, D.. 1988. Embryonic temperature determines adult sexuality in a reptile. Nature,
322: 832-834.
Hairston, C.S. and Burchfield, P.M., 1992. The reproduction and husbandry of the water monitor Vurunu.!
sulvutor. Int. Zoo Yearb., 3 1: 124- 130.
Hammack, S.H., 1989. Reproduction of the Colombian milk snake (Lumpropehis triungulum untie.\iunu) at
the Dallas Zoo. Int. Zoo Yearb., 28: 172- 177.
Hawkins, J.R., 1993. The SRY gene. Trends Endwrinol. Metab., 4: 328-332.
Horn, H-G. and Visser, G.J., 1989. Review of reproduction of monitor lizards Vurunu.\ spp. Int. Zoo Yearb.,
28: 140-150.
Janzen, F.J., 1993. The influence of incubation temperature and family on eggs, embryos, and hatchlings of the
smooth softshell turtle Apulone muticu. Physiol. Zool., 66: 349-373.
Janzen, F.J., 1995. Experimental evidence for the evolutionary significance of temperature-dependent sex
determination. Evolution, 49: 864-873.
Janzen. F.J. and Paukstis, G.L., 1991a. Environmental sex determination in reptiles: ecology, evolution, and
experimental design. Q. Rev. Biol., 66: 149-179.
Janzen, F.J. and Paukstis, G.L., 1991b. A preliminary test of the adaptive significance of environmental sex
determination in reptiles. Evolution, 45: 435-440,
Joanen, T. and McNease, L.L.. 1989. Ecology and physiology of nesting and early development of the
american alligator. Am. Zool.. 29: 987-998.
Johnson, C.M., Bamett, M. and Sharpe, P.T.. 1995. The molecular biology of temperature-dependent sex
determination. Phil. Trans. R. Soc. London, Ser. B, 350: 987-998.
Jost, A,, 1961. The role of fetal hormones in prenatal development. Harvey Lect.. 55: 201-226.
Korpelainen, H., 1990. Sex ratios and conditions required for environmental sex determination in animals.
Biol. Rev. Camb. Phil. Soc., 65: 147- 184.
Lance, V.A. and Bogart, M.H., 1994. Studies on sex determination in the american alligator Alli~ulor
mi.s.sis.sippiensi.-.. J. Exp. Zool., 270: 79-85.
Lang, J.W. and Andrews, H.V., 1994. Temperature-dependent sex determination in crocodilians. J. Exp. Zool.,
270: 28-44.
Lang, J.W., Andrews, H. and Whitaker. R.. 1989. Sex determination and sex ratios in Crocodylus, pulu-iiri.\.
Am. Zool., 29: 935-952.
Mardon, G., Mosher, R., Disteche. C.M., Nishioka, Y., McLaren. A. and Page, D.C., 1989. Duplication.
deletion and polymorphism in the sex-determining region of the mouse chromosome. Science, 243: 78-83.
McLaren. A,, 1988. Sex determination in mammals. Trend Genet., 4: 153-156.
Nevo, E., Beile, A. and Ben-Shlomo, R., 1984. The evolutionary significance of genetic diversity: ecological,
demographic and life history correlates. Lee. Notes Biomath.. 5 3 13-213.
Nichols, J.D. and Chabreck, R.H., 1980. On the variability of alligator sex ratios. Am. Nat., 116: 125-137.
Olmo, E., 1986. A. Reptilia. In: B. John (Editor), Animal Cytogenetics, Vol. 4: Chordata 3. Gebmder
Borntraeger, Berlin, pp. 1 - 100.
Osgood, D., 1980. Sex ratio and incubation temperature in a watersnake. Q. Rev. Biol., 55: 21.
Packard, G.C. and Packard. M.J.. 1988. The physiological ecology of reptilian eggs and embryos. In: C. Gans
and R.B. Huey (Editors). Biology of the Reptilia, Vol. 16. Alan R. Liss, New York, pp. 523-605.
Palmerallen. M., Beynon, F. and Georges, A., 1991. Hatchling sex-ratios are independent of temperature in
field nests of the long-necked turtle, Chelnilinu lon~colli. \ (Testudinata, Chelidae). Wildl. Res., 18:
225-23 1.
Pieau, C., 1971. Sur la proportion sexuelle chex les embryons de deux Cheloniens (Testudo gruecu L. et 'my.
~rbiculuri.\ L.); issues d'oeufs incubes artificiellement. C.R. Acad. Sci. Paris. 272 (D): 3071-3074.
Pieau, C.. 1972. Effects de la temperature sur Ie developpement des glandes genitales chez les embryons de
deux Cheloniens, Emy orbituluris L. et Teslucio gruecu L. C.R. Acad. Sci. Paris, 274 (D): 719-722.
Pieau, C.. 1974. Differenciation du sexe en fonction de la temperature chez les embryons d'Emy.\ orbi i ul ur~. ~
L. (Chelonien). effects des hormones sexuelles. Ann. Embryol. Morphogenet., 7: 365-394.
Pieau, C., 1976. Donnees recentes sur la differenciation sexuelle en fonction de la temperature chez les
embryons d'Emy, orbnuluris L. (Chelonien). Bull. Sw. Zool. Fr. Suppl., 4: 46-53.
Pieau, C., 1982. Modalities of the action of temperature on sexual differentiation in field-developing embryos
of the European pond turtle Emyf. orbicu1uri.s (Emydidaej. J. Exp. Zool., 220: 353-360.
264 C. Cioji. 1.R. Su~ingIuiul/Applied Animal Behuviour Science 51 11997) 251-265
Pieau, C.. 1996. Temperature variation and sex determination in reptiles. BioEssays, 18- 19-26.
Pieau, C. and Dorizzi, M., 1981. Determination of temperature sensitive stages for sexual differentiation of the
gonads in embryos of the turtle, Emys orhiruluris. J. Morphol., 170: 373-382.
Pieau, C., Girondot, M.. Richardmercier, N., Desvages, G., Dorizzi. M. and Zaborski, P., 1994a. Temperature
sensitivity of sexual-differentiation of gonads in the European pond turtle: hormonal involvement. J. Exp.
Zool., 270: 86-94.
Pieau, C., Cirondot, M., Desvages. G., Dorizzi, M.. Richard-Mercier. N. and Zaborski, P., 1994b. Environmen-
tal control of gonadal differentiation. In: R.V. Short and E. Balaban (Editors), The Differences between the
Sexes. Cambridge University Press, pp. 433-448.
Raynaud, A. and Pieau, C.. 1985. Environmental control of gonadal differentiation. In C. Gans and F. Billet
(Editors), Biology of the Reptilia, Vol. 15. John Wiley. New York, pp. 149-300.
Rigaud, T., Juchault, P. and Mocquard, J.P.. 1991. Experimental study of temperature effects on the sex ratio
of broods in terrestrial crustacea Armudillum vul t ure: possible implications in natural populations. J. Evol.
Biol., 4: 603-617.
Servan, J., Zaborski, P., Dorizzi, M. and Pieau, C., 1989. Female-biased sex ratio in adults of the turtle Emys
orhk'uluris at the northern limit of its distribution in France: a probable consequence of interaction of
temperature with genotypic sex determination. Can. J. Zool.. 67: 1279- 1284.
Smith, C.A., Elf, P.K., Lang, J.W. and Joss, J.M.P., 1995. Aromatase enzyme activity during gonadal sex
differentiation in Alligator embryos. Differentiation, 58: 28 1-290.
Smith, M.H. and Scribner, K.T., 1990. Population genetics of the slider turtle. In. J.W. Gibbons (Editor), Life
History and Ecology of the Slider Turtle. Smithsonian Institute Press, Washington DC, pp. 74-81.
Spotila, J.R., Spotila, L.D. and Kaufer, N.F., 1994. Molecular mechanisms of TSD in reptiles: a search for the
magic bullet. J. Exp. Zool., 270 1 17- 127.
Thomas, E.O., Licht, P.. Wibbels, T. and Crews. D.. 1992. Hydroxysteroid dehydrogenase activity associated
with sexual differentiation in embryos of the turtle Truiheniys .icr~pra Biol. Reprod., 46: 140-145.
Tiersch, T.R., Mitchell. M.S. and Wachtel, S.S., 1991. Studies on the phylogenetic conservation of the SRY
gene. Hum. Genet., 87: 571 - 173.
Tousignant, A. and Crews, D., 1995. Incubation-temperature and gonadal sex affect growth and physiology in
the leopard gecko (Eubl ephurf, muculuriu.~), a lizard with temperature-dependent sex determination. J.
Morphol., 224: 159- 170.
Valleley, E.M.A., Muller. U. , Ferguson. M.W.J. and Sharpe. P.T.. 1992. Cloning and expression analysis of 2
ZFY-related zinc finger genes from Alli,yuror mi.s.vI.s\;ppiensi.~, a species with temperature dependent sex
determination. Gene, 1 19: 221 -228.
Viets, B.E., Ewert, M.A., Talent, L.G. and Nelson, C.E.. 1994. Sex-determining mechanisms in squamate
reptiles. J. Exp. Zool., 270: 45-56.
Wachtel, S.S., 1993. Molecular Genetics of Sex Determination. Academic Press. San Diego, CA.
Webb, G.J.W., Beal, A.M., Manolis, S.C. and Dempsey, K.E., 1987. The effect of incubation temperature on
sex determination and embryonic development rate of Crococly1u.s johnsioni and C. por o. ~u. ~. In: G.J.
Webb, S.C. Manolis and P.J. Whitehead (Editors), Wildlife Management: Crocodiles and Alligators.
Surrey Beatty and Sons, Chipping Norton, UK, pp. 507-531.
Wibbels, T. and Crews, D., 1992. Specificity of steroid hormone-induced sex determination in a turtle. J.
Endocrinol., 133: 12 1 - 129.
Wibbels, T. and Crews, D., 1994. Putative aromalase inhibitor induces male sex determination in a female
unisexual lizard and in a turtle temperature-dependent sex determination. J. Endocrinol., 141: 295-299.
Wibbels, T., Bull, J.J. and Crews, D., 1991. Synergism between temperature and estradiol: a common pathway
in turtle sex determination. J. Exp. Zool., 260: 130- 134.
Wibbels, T., Bull, J J . and Crews, D.. 1994. Temperature-dependent sex determination: a mechanistic
approach. J. Exp. Zool., 270: 71-78.
Wilbur, H.M. and Morin. P.J., 1988. Life history evolution in turtles. I n C. Gans and R.B. Huey (Editors),
Biology of the Reptilia, Vol. 16. Alan R. Liss. New York, pp. 387-440.
Woodward, D.E. and Murray. J.D., 1993. On the effect of temperature dependent sex determination on sex
ratio and survivorship in crocodilians. Proc. R. Soc. London, Ser. B. 252 149- 155.
Ynetema, C.L., 1976. Effects of incubation temperatures on sexual differentiation in the turtle. Chelydru
.\erpenfinu. J. Morphol., 150: 453-462.
C. do$. I. R. Sw~nylund /Applied Animul Bfliuviour Stlence 51 (1997) 251 -265 265
Zaborski, P., Dorizzi, M. and Pieau, C.. 1982. H-Y antigen expression in temperature sex-reversed turtles
(Emyii orhiculuri.\). Differentiation, 22: 73-78.
Zaborski, P.. Dorizzi, M. and Pieau, C. , 1988. Temperature-dependent gonadal differentiation in the turtle
Emys orhiculuris: concordance between sexual phenotype and serological H-Y antigen expression at
threshold temperature. Differentiation, 38. 17-20.