Temperature-dependent Sex Determination of a Sea Turde, Caretta ...

Bull. Kitakyushu Mus. Nat. Hist., 18: 147-156. March 31, 1999
Temperature-dependent Sex Determination of a Sea Turde,
Caretta caretta, from Miyazaki,Japan
Shoji Tokunaga1, Yuji Iwakiri2* and Yoshito Nakajima3
'Department of Public Health, School ofMedicine, Kyushu University, Fukuoka 812-82,Japan
2Laboratory of Reproductive Physiology, Department of Fisheries, Kagoshima University,
Shimoarata 4-50-20, Kagoshima 890,Japan
'The Association forWildlife Research at Miyazaki, Ooaza-Shimanouchi 9779-8,
Miyazaki880-01,Japan
(Received November 14, 1998)
Abstract Eggs of Caretta caretta collected from Miyazaki, Japan, were incubated at
constant temperatures and a fluctuating temperature. A temperature shift experiment
was also conducted. Three clutches, comprising 332 eggs in total, were used for the
experiments. The mean hatching successwas0.73. The incubation period was Fitted
to the equation, D = 1265.56164-78.04757 T+1.25232 T2 (r2 = 0.951), where D is
incubation duration (days) and T is the incubation temperature (°C). In all experi
ments, all hatchlings were clearlyone sex or the other. At constant temperatures, the
sex ratio (proportion of males) was 1.0 at 27.7°C, 0.52 at 28.7°C, 0.51 at 29.7°C and 0.00
at 30.7°C. Bythe probit regression, the estimated critical temperature, the incubation
temperature giving 50% males, was 29.4°C and the estimated critical duration, the
incubation duration giving50% males, was54.7 days. At the fluctuating temperature,
27.7 (12h) and 30.7°C (12h), the sex ratio was 0.53. In the temperature shift experi
ment, the incubation temperature was27.7°Cfor 19daysand then wasshifted to 30.7°C.
The obtained sex ratio, 0.11,was closeto the sexratioexpected at the mean temperature
weighted by the developmental speed.
Key Words Temperature-dependent sex determination; Caretta caretta; sea turde; sex
ratio; critical temperature
Introduction
Sexdetermination of manyreptilesdepends on the temperature during incuba
tion ofthe eggs (reviews: Bull, 1980; Raynaud and Pieau, 1985; Korpelainen, 1990;
Paukstis andjANZEN, 1990; EwERTand Nelson, 1991;Janzen and Paukstis, 1991).
The significance oftemperature-dependent sex determination (TSD) in the fields of
evolutionary biology, ecology as well as conservation biology has been repeatedly
emphasized (Bull, 1980; Mrosovsky and Yntema, 1980; Mrosovsky, 1982; Bull,
1983; Bull, 1985;Bull and Charnov, 1989;Janzen and Paukstis, 1991).
Probably all sea turtles have TSD (Limpus et aL, 1985;Janzen and Paukstis,
* Presentaddressof YI: Wakita-apartment, 7-6 Sendou-machi, Saeki, Ooita 876,Japan

148 Shoji Tokunaga, Yuji Iwakiri and Yoshito Nakajima
1991). For sea turdes, high temperatures result in females and low temperatures
result in males. The temperature which gives 50% of each sex is called the critical
temperature (Pieau, 1976), the pivotal temperature (Mrosovsky and Yntema,
1980), the threshold temperature (Bull, 1980), or SDT50 (Limpus et al, 1985). In
this paper, the critical temperature was used to show the temperature giving 50% of
each sex, because this was the term first used to represent that temperature.
There have been some studies on TSD of Caretta caretta in the U.S.A. (Yntema
and Mrosovsky, 1980; Yntema and Mrosovsky, 1982; Mrosovsky, 1988) and
Australia (Limpus etal, 1985), but only a few reports are available on TSD inJapanese
populations of C. caretta and these were written in Japanese and published in a local
journal. Tokunaga (1991) first reported sex determination ofJapanese sea turtles
under constant temperatures. Asa preliminary report ofthis study, he reported that
nine hatched loggerheads from those incubated at 31°C (no correction for evapora
tive cooling) were females. Kamezaki and Kuroyanagi (1991) incubated one
clutch ofeggs from Atsumi, Aichi,Japan and reported that the sex ratio (number of
hatched turtles, hatching rate) of hatched turdes was 1.0 (34, 0.87) at 26°C, 1.0 (35,
0.90) at 28°C and 0.0 (37,0.95) at 30°C. He concluded that the critical temperature
of the clutch of C. caretta exists between 28 and 30°C.
This paper deals with TSD of C. caretta from Miyazaki, Japan. The critical
temperature of the loggerhead from Miyazaki and other localities in Japan, as well,
has not been identified so far. This study also intended to examine sex determina
tion underfluctuating temperature. The possible influence ofTSD on the hatchling
sex ratio is discussed.
Materials and methods
Eggs were collected from Miyazaki,Japan. The geography and the turtle nest
ing habitat of the beach have been described by Iwamoto etal (1985).
Experiments were conducted at the Laboratory of Ecology, Faculty of Science,
Kyushu University, in 1989. Three clutches were used for the experiments. Each
clutch was removed from its nest by YN after the turtle completed laying and was
packed in a Styrofoam box for transport. The eggs were sent to the laboratory by
commercial truck delivery with no attempt to control the temperature of the eggs.
The number of eggs, date of oviposition and the date of starting incubation ofeach
clutch were 127,June 30 and July 4 for the clutch A; 135,July 2 and July 7 for the
clutch B; 138,July 2 andJuly 7 for the clutch C.
Each clutch was divided into three to five groups (see Table 1). Each group of
eggswasincubated in a plasticcontainer (approx. 17 x 31 cm on the bottom) in two
layers and the bottom of the container was coveredwithwet vermiculite. A layerof
eggs, usually 28 eggs, was set on the vermiculite and covered with a mesh to keep air
spacebetween the eggs. Wetvermiculite was put on the mesh,and the secondlayer

Sex determination of a sea turtle 149
of eggs was set, covered with a mesh and a third layer of wet vermiculite. Each
plastic containerwas covered with a sheet ofaluminium foil with holes for ventilation.
The eggs were incubated in SANYO MIR-150E incubators or Shimadzu BITEC
300 Low-Temp incubators. The air in each incubator was constandy circulated by
an interior fan to prevent a temperature gradient. The temperature of each incuba
tor wasmonitored dailywith an alcohol thermometer approximately at the middle of
the incubator air space. The thermometers had been calibrated againsta standard
thermometer before the experiment. Temperature was also monitored by the
standardthermometeroncea week. It was confirmed that temperaturedid not vary
excessively from the designed temperature by daily reading maximum-minimum
thermometer which were set in each incubator. The preliminary experiment
showed that the temperature in the wetvermiculite was cooler than the air tempera
ture by 0.3°C due to evaporative cooling. Therefore the incubation temperatures
were corrected by 0.3°C from the air temperature inside the incubators.
The incubation at fluctuating temperature was carried out using a program
mable BITEC 300 incubator. The temperature was fluctuated from 27.7 for 12
hours to 30.7°C for 12 hours daily. In the temperature shift experiment, the eggs
were incubated at 27.7°C for 19 days and then shifted to 30.7°C for the remainder of
the incubation period.
Adish ofwater was setin eachincubator to moisten the air. Water was sprayed
on the vermiculite when signs of desiccation were observed on the surface of
the vermiculite. The humidity of each incubator was monitored daily with a
simple psychrometer. The humidity was always >80%, usually >90%. No major
differences were detected between incubators.
The dateof hatching was defined asthe day when the whole body emerged out
side the egg. Hatchlings were killed by injection of about lOmg of Pentobarbital
Sodium Salt and fixed in 10% buffered formalin solution after the body cavity was
opened. The left gonad, oviductand attached kidneywere removed. The tissues
were embedded in Paraplast and cut in 4-8 /xm sections which were stained with
haematoxylin and eosinor haematoxylin and the periodicacid-Schiff reaction (PAS);
the latter gave better results.
All specimens were microscopically sexed using the following histological
criteria. Withmalesthe medullawas differentiated into seminiferous tubules,while
the cortex was reduced. With females the cortex was well developed and the
medulla showed no development of seminiferous tubules. Following sexing of a
portion of the specimens byST, mostspecimens were sexed byYI. YI reexamined
the sexing ofall specimens at least twice.
Statistical calculations were conducted by SAS, SAS Institute (Cary, NC) at the
Computer Center, Kyushu University, except the Fisher's exact test and 95% confi
dence intervals oftheproportion which were calculated by Stata (Stata Corp.). The
level of statistical significance (a) is 0.05.

150 Shoji Tokunaga, Yuji Iwakiri and Yoshito Nakajima
Results
Table 1 shows hatching success, incubation duration and sex ratio of each
incubation group. In all experiments, all hatchlings were clearly one sex or the
other. The overall mean (SE, range) hatching success of the 11 groups was 0.73
(0.036, 0.54- 0.93). Although the variation of hatching successamong clutches was
not significant, the difference of hatching success among constant incubation
temperatures was significant. The hatching success at 29.7°C was significantly
higher than those at 27.7°C (P30.7d 32c 0.65 55.0(0.14,54-56) 4 16 0 0.20(0.06-0.44)
C2 27.730.7f 56 0.84* 55.1 (0.11,54-57) 22 25 0 0.47(0.32-0.62)
C2 27.730.7f 25 0.76 55.4(0.22,54-58) 13 6 0 0.68(0.43-0.87)
a: Incubationtemperatureaftera -0.3°C correction forevaporative cooling.
b: Sexratio isproportion of males, andits95% confidence intervals were calculated bythe exact
method,
c: Unsexable because oflogistic problems.
d: Incubation temperature was initially 27.7°C andshifted to 30.7°C during incubation,
e: One egg was removed during incubation.
f: Incubation temperaturewas fluctuated from 27.7°C (12h) to 30.7°C (12h) everyday.
g: One pair of dead Siamese twins was found in an egg (unsexed and eliminated from further
analyses).
For the constanttemperature experiment, the relationship between incubation
duration (D) and incubation temperature (T) was fitted to the quadratic equation:
D = 1265.56164-78.04757 •T+1.25232 •T2 (r2= 0.951).
Figure 1 shows the sex ratios of the hatched turtles incubated at constant

Sex determination of a sea turtle
151
28 29 30
Temperature (°C)
Fig.1. Constant incubation temperature and sexratio (proportion of males) of Caretta
caretta from Miyazaki,Japan. The bar on the solid circle (sex ratio) shows the
95% confidence intervals of sex ratio calculated by the exact method assuming
binomial distribution. The curve is fitted by the probit regression using SAS.
The sex ratios of two groups incubated at 29.7°C are shown separately, because
their sex ratios are significantly different (two-tailed Fisher's exact test).
31
temperatures. The variation of the sex ratios of two incubation groups at 27.7°C
and 30.7°C was not significant, but the sex ratios of the two incubation groups at
29.7°C were significantly different (two-tailed Fisher's exact test). Hence the sex
ratios were separatelyshown for the two groups incubated at 29.7°C. The sex ratio
of hatched turtles was highly dependent on incubation temperature. The sex ratio
(95% confidence intervals by the exact method) was 1.0 (0.89-1.0) at 27.7°C,
0.52 (0.31-0.73) at 28.7°C, 0.13 (0.03-0.34) and 0.85 (0.65-0.96) at 29.7°C and 0.0
(0.0-0.10) at 30.7°C. The relationship between incubation temperature and sex
ratio was fitted to the probitcurve. By the probit regression, the critical tempera
ture, the incubation temperature that should result in 50% males, was calculated to
be 29.4°C. The critical duration, the incubation duration that should result in 50%
males, was also calculated by fitting the relationship between the incubation period
of each incubation group and the sex ratio to the probit curve. By the probit
regression, the critical duration was calculated to be 54.7 days.
At the fluctuating temperature, there was no significant differencebetween the
sex ratios of the two incubation groups, C2 and C3 (two-tailed Fisher's exact test).
The overall sex ratio (95% CI) at the fluctuating temperature was 0.53 (0.40-0.65).
By theabove probit regression ofsex ratio against constant temperature, theexpected
sexratio at the constant temperature of 29.2°C (thecrudemean temperature of the
fluctuating temperature) was 0.57. When the difference ofdevelopmental speed by
incubation temperature was considered, the mean temperature was calculated to be
29.4°C, at which temperature the expected sex ratio was 0.48.

152 Shoji Tokunaga, Yuji Iwakiri and Yoshito Nakajima
The two incubation groups (B5 and CI) of the temperature shift experiment
showed no significant difference in sex ratio. The overallsex ratio (95% CI) by the
temperature shift experiment was 0.11 (0.03-0.25). Considering only the duration
of incubation period at each temperature, the mean incubation temperature was
calculated to be 29.7°C,at which temperature the expected sex ratio was0.35. When
the difference of developmental speed by incubation temperature was also consid
ered, the weighted mean incubation temperature was 30.1°C, and the expected sex
ratio was 0.20.
Discussion
In this series of experiments, all eggswere kept at uncontrolled temperature for
4 or 5 daysduring transportation. If the uncontrolled temperature during transpor
tation affected sex determination, interpretation of the present results on sex deter
mination would be difficult. However, the presence of the critical period among
TSD species should be noted. Before and after this critical period, sex determina
tion is insensitive to temperature. This was demonstrated in an experiment on
the critical period of sex differentiation in whichYntema and Mrosovsky (1982)
temporarily alternated the incubation temperature either upwards or downwards
from the critical temperature. Theyshowed that the incubation temperature in the
first 11 days of incubation had no effecton sexual differentiation of the loggerhead
sea turde. Therefore, the results of the present studyare not likelyto be attributable
to temperatures experienced prior to the controlled incubation.
The temperatures during transportation might have affected the incubation
period. Consequently, both the relationship between incubation temperature and
incubation period and the critical period of incubation might have some degree of
error. However, the duration under uncontrolled temperature waslessthan 10% of
the total incubation period. Furthermore, the difference of incubation tempera
ture changes the incubation duration by 24% (from 27.7 to 30.7°C, thisstudy) or by
44% (from 25 to 32°C,Limpus etal, 1985). This means that even if the temperature
during transportation differed by 7°C among clutches, the overall incubation
duration was affected bylessthan 5%. Thus, the effectof uncontrolled temperature
duringtransportation on incubation duration should have beenwithin the allowable
margin of error.
The critical temperature has been reported for C. caretta from some localities.
Limpus et al (1985) examined the critical temperature of C. caretta from Heron
Island and Mon Repos, Australia. They estimated that the critical temperature with
95% confidence limits for the combined clutches from the two localities was 28.6 ±
0.5°C. Mrosovsky (1988) incubated the eggs from North Carolina, Georgia and
Florida, USA. He estimated the critical temperature of the eggs from the three
localities combined was 29.0°C. Kamezaki and Kuroyanagi (1991) estimated that

Sex determination of a sea turtle 153
the critical temperature ofa clutch of C. caretta from Atsumi, Japan exists between 28
and 30°C. The present estimate of critical temperature was 29.4°C, which is in the
range of the critical temperature reported for the clutch from Atsumi. It was close
to the overall estimate of 28.6°C given by Limpus et al (1985) and 29.0°C given by
Mrosovsky (1988).
In some turdes with TSD, dominance of one-sex-determining temperature
over another-sex-determining temperature is known. In map turtles {Graptemys
ouachitensis), for example, an isolated pulse ofmale-producing temperature can have
a substantial male-inducing effect, although an isolated pulse of female-producing
temperature in the sensitive period has litde female-producing effect (Bull and
Vogt, 1981). Thus, sex is more readily influenced by male-determining tempera
ture than female-determining temperature in this species. In snapping turtle
(Chelydra serpentina), on the contrary, female determination is more easily induced
than male determination. Incubation at female-producing temperature through
either halfofthe sensitive period induces femaleness in most embryos; male determi
nation requires incubation at male-producing temperature for nearly the entire
duration of the sensitive period (Yntema, 1979). In this study, the sex ratio at
fluctuating temperature was close to the expected sex ratio at crude mean tempera
ture (0.53 vs. 0.57) and that at weighted mean temperature considering the develop
mental speed (0.53 vs. 0.48), suggesting a lack of dominance ofone-sex-determining
temperature over another-sex-determining temperature in C. caretta.
The present study, along with other studies on sex determination of C. caretta,
shows that the sex ratio of the hatchling turdes can vary in response to subde differ
ences in the thermal environment of the eggs. Hence, a variety of environmental
factors of the nest can cause significant differences in the hatchling sex ratio of C.
caretta. For example, the brown sands of Mon Repos are consistendy warmer than
the white sands of Heron Island. The hatchling sex ratio at Mon Repos was biased
toward females by about 30% compared to that at Heron Island (Limpus et al,
1983). Seasonal change of hatchling sex ratio was also observed. Sex ratios of
hatchling C. caretta taken from South Carolina and Georgia ranged from no females
in nests laid in late May to 80% females in those laid in early July; the sex ratio
decreased to 10% females in nests laid in early August (Mrosovsky etal, 1984). It
has also been demonstrated that the hatchling sex ratios of C. caretta varygeographi
cally. The overall hatchling sex ratio produced in South Carolina and Georgia from
1979 to 1982 was estimated to be 0.437 (Mrosovsky etal, 1984). At Florida beach,
the estimated sex ratios (year) of hatchling turdes were 0.033-0.074 (1986), 0.001-
0.053 (1987) and 0.11-0.13 (1988), and strongly female-biased sex ratios were
expected in 1989 and 1990, as well, by the observed high sand temperatures
(Mrosovsky and Provencha, 1992). These highly female-biased sex ratios are
noteworthy, because 90% of U.S. C. caretta were estimated to nest in Florida
(Murphy and Hopkins, 1984).

154 Shoji Tokunaga, Yuji Iwakiri and Yoshito Nakajima
There are only a few reports on hatchling sex ratio of Japanese C. caretta.
Kuroyanagi and Kamezaki (1993) examined the hatchling sex ratio at Atsumi in
1991. The sex ratios (date of oviposition) of randomly selected 30 hatchlings for
each clutch were 0.900 (June 7), 0.607 (June 20), 0.900 (July 7), 0.759 (July 16),
0.333 (July 30) and 0.379 (August 7), suggesting a seasonal influence on hatchling
sex ratio. Iwakiri (unpublished) observed hatchling sex ratio at Fukiagehama,
Kagoshima in 1991. The hatchling sex ratio (date of oviposition) of randomly se
lected 30 hatchlings for each clutch were 1.00 (May 26), 0.20 (June 6), 0.77 (June
19), 0.57 (June 29), 0.00 (July 13) and 0.23 (July 26). Combined with the data on
nesting frequency in the beach, he estimated that the overall sex ratio of hatched
turtles in the bay was 0.55. The results of these studies suggest that the seasonal
variation of hatchling C. caretta sex ratio exists also in Japan.
Although a geographic comparison of hatchling sex ratio has not been done in
Japan, geographical variation of hatchling sex ratio can be estimated by a study of
sand temperatures in 1993. Matsuzawa and Sakamoto (1994) measured sand
temperature at major nesting beaches ofJapanese sea turtles by data logger MDST at
a depth of either 40cm or 60cm or both from April to November in 1993. The
observed sand temperature showed that sand temperatures were higher than the
critical temperature, 29.4°C, for 10 or fewer days in the nine nesting beaches, except
two which were the most southern beaches. In C.caretta, females were not produced
when the temperature exceeded the critical temperature for 10 days or less (Yntema
and Mrosovsky, 1982). Consequently, it is suggested that female hatchlings were
not produced in mostJapanese nesting beaches in 1993. However, it is noted that
the weather conditions in 1993 were exceptionally rainy and cool. More females are
expected to be produced under the weather conditions occurring in an average year.
Therefore, Japanese C. caretta populations, like those in other areas, are prob
ably subjected to a bias and variation of hatchling sex ratio due to geographic
location and seasonal and yearlyweather conditions as C. caretta in other areas. The
bias and variation ofsex ratio, if present, apparently contradict the theoretical predic
tion on sex ratio evolution. Fisher (1930) predicted that if the parental investment
needed to produce a male ora female is equal, strong frequency-dependent selection
against skewed sex ratio works and the sex ratio ofhatchlings should be close to 1:1 at
the time when parental investment ends. The population of C. caretta in the north
western Pacific is known to nests only in Japanese islands (Kamezaki, personal
com.). It is important to examine the presence or absence of the bias and variation
of the hatchling sex ratio and, if they present, their degree in the Japanese
population. Surveysof hatchling sex ratio ofJapanese C. caretta over a long period
and covering the all range of the nesting beaches are desirable.

Sex determination of a sea turtle 155
Acknowledgments
Dr Y. Ono and members of the laboratory of Ecology, Faculty ofScience, Kyushu
University encouraged and helped the author(ST) during incubation experiments.
We are grateful to Drs C. Limpus and P. Harlow for valuable comments on the
manuscript and to Mis L. Fillipi for linguistic advice. We thank Mr H. Suganuma
and Dr C.J. Limpus for invaluable advice on the method of incubation. The Asso
ciation for Wildlife Research at Miyazaki helped with the collection and the delivery
of turde eggs. This study was supported by the 15th Nissan Fund.
References
Bull,J. J. 1980. Sex determination in reptiles. Quarterly Review ofBiology, 55: 3-21.
Bull,J. J. 1983. Evolution ofSexDetermining Mechanisms, Benjamin/Cummings, London.
Bull,J. J. 1985. Sex determining mechanisms: an evolutionary perspective. Experientia, 41: 1285-
1296.
Bull,J.J. and E. L. Charnov. 1989. Enigmatic reptilian sex ratios. Evolution, 43: 1561-1566.
Bull, J. J. and R. C. Vogt. 1981. Temperature-sensitive periods of sex determination in emydid
turtles, fournal ofExperimental Zoology, 218: 435-440.
Ewert, M. A. and C. E. Nelson. 1991. Sex determination in turtles: diverse patterns and some
possible adaptive values. Copeia 1991:50-69.
Fisher, R. A. 1930. TheGenetical Theory ofNaturalSelection, Oxford University Press.
Iwamoto, T., M. Ishii, Y. Nakashima, H. Takeshita and A. Itoh. 1985. Nesting cycles and migra
tions of the loggerhead sea turde in Miyazaki,Japan. Japanesefournal ofEcology, 35: 505-511.
Janzen, F.J. and G. L. Paukstis. 1991. Environmental sex determination in reptiles: ecology,
evolution, and experimental design. Quarterly Review ofBiology, 66: 149-179.
Janzen, F.J. and G. L. Paukstis. 1991. A preliminary test of the adaptive significance of environ
mental sex determination in reptiles. Evolution, 45: 435-441.
Kamezaki, N. and K. Kuroyanagi. 1991. On the TSD of Caretta caretta from Japan. Umigame
Newsletter ofJapan, 7: 8-10. (in Japanese)
Korpelainen, H. 1990. Sex ratios and conditions required for environmental sex determination in
animals. Biological Review, 65: 147-184.
Kuroyanagi, K. and N. Kamezaki. 1993. The sex ratio of Caretta caretta hatchlings at Atsumi
peninsula and the possibility of predicting hatchling sex ratio. Umigame Newsletter ofJapan,
15: 20-21. (Abstract, in Japanese)
Limpus, C. J., P. Reed and J. D. Miller. 1983. Islands and turtles. The influence of choice of
nesting beach on sex ratio. In BakerJ.T., R. M. Carter, P. W. Sammarco and K. P. Stark
(eds.), Proceedings: Inaugural Great Barrier Reef Conference, pp. 397-402, James Cook University
Press, Townsville.
Limpus, C. J., P. Reed and J. D. Miller. 1985.
Temperature dependent sex determination in
Queensland sea turtles: intraspecific variation in Caretta caretta. In Grigg, G., R. Shine and H.
Ehmann (eds.), Biology ofAustralasian Frogs andReptiles, pp. 343-351, Royal ZoologicalSociety,
New South Wales.
Matsuzawa, Y. and W.Sakamoto. 1994. Seasonalfluctuation of sand temperature at the major sea
turtle rookeries injapan during the 1993season. Umigame Newsletter ofJapan, 21:9-13. (inJapa
nese)

156 ShojiTokunaga, Yuji Iwakiri and Yoshito Nakajima
Mrosovsky, N. 1982. Sex ratio bias in hatchling sea turtles from artificially incubated eggs.
Biological Conservation, 23: 309-314.
Mrosovsky, N. 1988. Pivotal temperatures for loggerhead turtles {Caretta caretta) from northern
and southern nesting beaches. CanadianJournal ofZoology, 66: 661-669.
Mrosovsky, N., S. R. Hopkins-Murphy and J. I. Richardson. 1984. Sex ratio of sea turtles:
seasonal changes. Science, 225: 739-741.
Mrosovsky, N. and J. Provancha. 1992. Sex ratio of hatchling loggerhead sea turtles: data and
estimates from a 5-yearstudy. CanadianJournal of Zoology, 70: 530-538.
Mrosovsky, N. and C. L. Yntema. 1980. Temperature dependence of sexual differendation in sea
turdes: implications for conservation practices. Biological Conservation, 18: 271-280.
Murphy, T. M.and S.R. Hopkins. 1984. Aerialand ground surveys of marine turde nesting beaches
in the southeast region. Report tothe U.S. National Marine Fisheries Service, National Oceanic and
Atmospheric Administration, Miami, FL.
Paukstis, G. L.and F.J.Janzen. 1990. Sexdetermination in reptiles: summary of effectsof constant
temperatures of incubation on sex ratios of offspring. Smithsonian Herpetological Information
Service, 83: 1-28.
Pieau, C. 1976. Donnees recentes sur la differendation sexuelle en fonction de la temperature chez
les embryons d'Emys orbicularis L. (Chelonien). Bulletin de la Societe zoologique de France.
Supplement, 4: 46-53.
Raynaud, A. and C. Pieau. 1985. Embryonic development of the genital system. In Gans, C. and
F. Billet (eds.), Biology of the Reptilia, Vol. 15, pp. 149-300, Wiley.
Tokunaga, S. 1991. A preliminary observation on temperature-dependent sex determination in
twospecies ofJapanese sea turdes. Umigame Newsletter ofJapan, 7:3-7. (inJapanese with English
summary)
Yntema, C. L. 1979. Temperature levels and periods of sex determination during incubation of
eggs of Chelydra serpentina. J. Morphol, 159: 17-27.
Yntema, C. L. and N, Mrosovsky. 1980. Sexual differentiation in hatchling loggerheads {Caretta
caretta) incubated at different controlled temperatures. Herpetologica, 36: 33-36.
Yntema, C. L. and N, Mrosovsky. 1982. Critical periods and pivotal temperatures for sexual
differentiation in loggerhead sea turtles. CanadianJournal ofZoology, 60: 1012-1016.