Abstract There are a remarkable variety of sex determination systems among different animal taxa. In most animals, sex is determined chromosomally. Although in an increasing number of animals sex determination has been found to be influenced primarily by the environment. Species with genotypic sex determination (GSD) have their sex determined at the time of fertilization, by genetic factors alone and those with environmental sex determination (ESD) have their sex determined by environmental factors that act after fertilization. Temperature-dependent Sex Determination (TSD), whereby the sex of the developing embryos depends on the temperature at which they develop is widespread in oviparous reptiles and occurs in all crocodilians, marine turtles and tuatara examined to date and is common in many freshwater turtles and lizards. SECTION ONE Temperature-dependent sex determination (TSD) was never expected to occur in viviparous reptiles, as thermoregulation by pregnant females would result in relatively stable gestation temperatures. Temperature-dependent sex determination and viviparity goes against all the basic assumptions that TSD occurs in oviparous reptiles where temperatures within a nest vary widely. However, skewed sex ratios as a result of incubation temperature indicated the possibility of TSD in the viviparous lizard Eulamprus tympanum. In my first experiments I show the first recorded case of a viviparous reptile with TSD. The developing embryos of the viviparous skink E. tympanum are subject to TSD, with gestation temperature having a highly significant effect on sex and warmer temperatures giving rise to male offspring (Chapter 1). Sex is fully determined at the time of birth and can be differentiated histologically into testes or ovaries (Chapter 2). The morphology and histological characteristics of the gonads of neonatal E. tympanum resulting from the treatment temperatures described in chapter 1 illustrate that sex in E. tympanum is easily distinguished at the time of birth and corresponds with the presence or absence of hemipenes. Males are histologically characterised by an elongated gonad consisting of seminiferous tubules with either no cortical epithelium or, if present at all, in a very thin band. If they are present, M�llerian ducts, showing signs of degeneration, are attached to the kidney by a shortened mesosalpinx. Females are histologically characterised by an irregularly shaped gonad consisting of a thick cortical epithelium that occasionally contains oocytes. The M�llerian ducts are obvious structures attached to the kidney by a fibrous mesosalpinx. The presence or absence of hemipenes is a reliable technique for determining sex in newborn E. tympanum. Sex determination is easiest to perform on neonates within the first few days of birth as hemipenes become increasingly difficult to evert as neonates age, however, with practice they are easily identified without full eversion. SECTION TWO The thermal biology of E. tympanum in the field is restricted by both the thermal properties of their habitat (Chapter 3) and behavioural modifications when faced with a predation threat (Chapter 4). The available temperatures in the field suggest that TSD is biologically relevant in the species and not just a laboratory artefact; E. tympanum can attain mean selected temperatures achieved in the laboratory but the proportion of time at the temperature is restricted. Females actively thermoregulate in the field, although they are restricted in their efficiency of thermoregulation by environmental constraints, for example, microhabitat structure, weather conditions, predator avoidance and social ranking. The highly territorial nature and high densities of E. tympanum present in Kanangra Boyd National Park potentially force less dominant individuals into less favourable habitats that are significantly cooler. An important point is that gravid females in more favourable habitats in the period encompassing the middle third of development (the assumed sex determining period) are selecting higher temperatures, with lower variance and have greater thermoregulatory efficiency than during the rest of pregnancy, therefore, thermoregulating more precisely during this thermosensitive period (Chapter 3). Chemosensory cues provide important information on the risk of predation. Hence, chemoreception is a common mechanism used by many species to detect the presence of, and subsequently respond to, a potential predator. The perceived risk of predation may force retreat to sub-optimal conditions, forcing a trade-off between the risk of predation and the ability to acquire resources. The basking regime maintained by gravid female E. tympanum, can directly alter sex ratios of offspring produced through temperature-dependent sex determination (Chapter 1). The avoidance of predator scents may restrict basking ability and in turn alter the sex of offspring produced. I measured responsiveness to chemical cues using tongue flicks as an indicator of chemical discrimination in females of different reproductive condition. I then measured activity and basking behaviour of gravid and non-gravid females in experimental enclosures in the presence of various chemical stimuli to determine if basking opportunity is compromised by the presence of a predator scent. Females respond differently depending upon reproductive condition, with gravid females responding most significantly to a predator scent. Activity, basking frequency, and time spent in the open (basking duration) are significantly reduced in gravid females in the presence of a predator stimulus. Under laboratory conditions, gravid females modify their behaviour and forego the opportunity to bask when there is a perceived predation risk (Chapter 4). SECTION THREE As female viviparous reptiles can regulate the temperature of the embryo by maternal temperature selection (Chapter 1), the occurrence of TSD in E. tympanum opens the possibility for females to select the sex of offspring. Reproducing females may benefit by facultatively adjusting their investment into sons over daughters or vice versa, in response to population wide shifts in adult sex ratios. Female E. tympanum, can manipulate the sex of their offspring in response to sex imbalances in the population using temperature-dependent sex determination (Chapter 5). When adult males are scarce, females produce male-biased litters and when adult males are common, females produce female-biased litters. The cues used by a female to assess the adult population are not known, but presumably depends upon the female�s experience throughout the breeding season and is the subject of further investigation (Chapter 6). The maternal manipulation of offspring sex ratio in E. tympanum suggests a selective advantage of temperature-dependent sex determination. Any facultative sex ratio response needs to recognise the scarcity of one sex in order to overproduce that sex in the next generation; offspring sex ratio will vary inversely with adult sex ratio. Maternal sex allocation in E. tympanum is linked with population (or adult) sex ratio (Chapter 5), and one of the mechanisms by which females recognise an imbalance may be linked to visual recognition of males (Chapter 6). Females maintained throughout pregnancy without any male stimulus produce entirely male offspring (Chapter 5). In contrast females exposed to male stimulus produce both sexes (Chapter 5). Females respond differently to varying degrees of male stimulus and visual recognition of males in a population may be more important than chemoreception. In the absence of visual cues, females produce more male offspring, even when chemosensory cues are present (Chapter 6). The study system presented here offers many advantages over oviparous species with TSD, due to E. tympanum being relatively short lived and fast maturing. Thus, the fitness consequences over multiple generations as a result of gestation can be investigated. Viviparity allows maternal control of embryonic temperature during gestation and a means of maternal sex allocation. Until now the maternal side of TSD and sex allocation has been where the mother deposits her eggs and the allocation of sex steroid hormones at oviposition, both of which have been difficult to study. The work presented and the study system itself should inspire great interest in TSD and viviparous reptiles.

Abstract There are a remarkable variety of sex determination systems among different animal taxa. In most animals, sex is determined chromosomally. Although in an increasing number of animals sex determination has been found to be influenced primarily by the environment. Species with genotypic sex determination (GSD) have their sex determined at the time of fertilization, by genetic factors alone and those with environmental sex determination (ESD) have their sex determined by environmental factors that act after fertilization. Temperature-dependent Sex Determination (TSD), whereby the sex of the developing embryos depends on the temperature at which they develop is widespread in oviparous reptiles and occurs in all crocodilians, marine turtles and tuatara examined to date and is common in many freshwater turtles and lizards. SECTION ONE Temperature-dependent sex determination (TSD) was never expected to occur in viviparous reptiles, as thermoregulation by pregnant females would result in relatively stable gestation temperatures. Temperature-dependent sex determination and viviparity goes against all the basic assumptions that TSD occurs in oviparous reptiles where temperatures within a nest vary widely. However, skewed sex ratios as a result of incubation temperature indicated the possibility of TSD in the viviparous lizard Eulamprus tympanum. In my first experiments I show the first recorded case of a viviparous reptile with TSD. The developing embryos of the viviparous skink E. tympanum are subject to TSD, with gestation temperature having a highly significant effect on sex and warmer temperatures giving rise to male offspring (Chapter 1). Sex is fully determined at the time of birth and can be differentiated histologically into testes or ovaries (Chapter 2). The morphology and histological characteristics of the gonads of neonatal E. tympanum resulting from the treatment temperatures described in chapter 1 illustrate that sex in E. tympanum is easily distinguished at the time of birth and corresponds with the presence or absence of hemipenes. Males are histologically characterised by an elongated gonad consisting of seminiferous tubules with either no cortical epithelium or, if present at all, in a very thin band. If they are present, M�llerian ducts, showing signs of degeneration, are attached to the kidney by a shortened mesosalpinx. Females are histologically characterised by an irregularly shaped gonad consisting of a thick cortical epithelium that occasionally contains oocytes. The M�llerian ducts are obvious structures attached to the kidney by a fibrous mesosalpinx. The presence or absence of hemipenes is a reliable technique for determining sex in newborn E. tympanum. Sex determination is easiest to perform on neonates within the first few days of birth as hemipenes become increasingly difficult to evert as neonates age, however, with practice they are easily identified without full eversion. SECTION TWO The thermal biology of E. tympanum in the field is restricted by both the thermal properties of their habitat (Chapter 3) and behavioural modifications when faced with a predation threat (Chapter 4). The available temperatures in the field suggest that TSD is biologically relevant in the species and not just a laboratory artefact; E. tympanum can attain mean selected temperatures achieved in the laboratory but the proportion of time at the temperature is restricted. Females actively thermoregulate in the field, although they are restricted in their efficiency of thermoregulation by environmental constraints, for example, microhabitat structure, weather conditions, predator avoidance and social ranking. The highly territorial nature and high densities of E. tympanum present in Kanangra Boyd National Park potentially force less dominant individuals into less favourable habitats that are significantly cooler. An important point is that gravid females in more favourable habitats in the period encompassing the middle third of development (the assumed sex determining period) are selecting higher temperatures, with lower variance and have greater thermoregulatory efficiency than during the rest of pregnancy, therefore, thermoregulating more precisely during this thermosensitive period (Chapter 3). Chemosensory cues provide important information on the risk of predation. Hence, chemoreception is a common mechanism used by many species to detect the presence of, and subsequently respond to, a potential predator. The perceived risk of predation may force retreat to sub-optimal conditions, forcing a trade-off between the risk of predation and the ability to acquire resources. The basking regime maintained by gravid female E. tympanum, can directly alter sex ratios of offspring produced through temperature-dependent sex determination (Chapter 1). The avoidance of predator scents may restrict basking ability and in turn alter the sex of offspring produced. I measured responsiveness to chemical cues using tongue flicks as an indicator of chemical discrimination in females of different reproductive condition. I then measured activity and basking behaviour of gravid and non-gravid females in experimental enclosures in the presence of various chemical stimuli to determine if basking opportunity is compromised by the presence of a predator scent. Females respond differently depending upon reproductive condition, with gravid females responding most significantly to a predator scent. Activity, basking frequency, and time spent in the open (basking duration) are significantly reduced in gravid females in the presence of a predator stimulus. Under laboratory conditions, gravid females modify their behaviour and forego the opportunity to bask when there is a perceived predation risk (Chapter 4). SECTION THREE As female viviparous reptiles can regulate the temperature of the embryo by maternal temperature selection (Chapter 1), the occurrence of TSD in E. tympanum opens the possibility for females to select the sex of offspring. Reproducing females may benefit by facultatively adjusting their investment into sons over daughters or vice versa, in response to population wide shifts in adult sex ratios. Female E. tympanum, can manipulate the sex of their offspring in response to sex imbalances in the population using temperature-dependent sex determination (Chapter 5). When adult males are scarce, females produce male-biased litters and when adult males are common, females produce female-biased litters. The cues used by a female to assess the adult population are not known, but presumably depends upon the female�s experience throughout the breeding season and is the subject of further investigation (Chapter 6). The maternal manipulation of offspring sex ratio in E. tympanum suggests a selective advantage of temperature-dependent sex determination. Any facultative sex ratio response needs to recognise the scarcity of one sex in order to overproduce that sex in the next generation; offspring sex ratio will vary inversely with adult sex ratio. Maternal sex allocation in E. tympanum is linked with population (or adult) sex ratio (Chapter 5), and one of the mechanisms by which females recognise an imbalance may be linked to visual recognition of males (Chapter 6). Females maintained throughout pregnancy without any male stimulus produce entirely male offspring (Chapter 5). In contrast females exposed to male stimulus produce both sexes (Chapter 5). Females respond differently to varying degrees of male stimulus and visual recognition of males in a population may be more important than chemoreception. In the absence of visual cues, females produce more male offspring, even when chemosensory cues are present (Chapter 6). The study system presented here offers many advantages over oviparous species with TSD, due to E. tympanum being relatively short lived and fast maturing. Thus, the fitness consequences over multiple generations as a result of gestation can be investigated. Viviparity allows maternal control of embryonic temperature during gestation and a means of maternal sex allocation. Until now the maternal side of TSD and sex allocation has been where the mother deposits her eggs and the allocation of sex steroid hormones at oviposition, both of which have been difficult to study. The work presented and the study system itself should inspire great interest in TSD and viviparous reptiles.

The leopard gecko (Eublepharis macularius) is a reptile species in which embryonic temperature contributes both to sex determination and within- sex polymorphisms. Its life history makes the leopard gecko a model system for seeking ontogenic and proximate explanations for within-sex variation in sexually dimorphic behavior and neurophysiology, necessary attributes for reproductive success. For my dissertation I have incorporated the role of androgens that potentially modulate incubation temperature effects on behavioral and brain variation, which I approached using embryo and adult leopard geckos. First, I found that that the bias of same-sex clutch siblings is primarily incubation temperature- dependent and any maternal or genetic effects on same-sex clutch siblings are secondary. Second, I found that testosterone concentrations in the yolk-albumen were higher in eggs of late development than early development at 26 °C, a female-producing incubation temperature, but did not differ from eggs incubated at another female-biased temperature. This increase in testosterone concentrations during the temperature sensitive period in putative females is a finding opposite of reported trends in most other reptiles studied to date. Further, I found that the embryonic environment influences male sociosexual investigation in the absence of gonadal hormones. Lastly, in adult males of 32.5 °C, a male-biased incubation temperature, I found that the phosphoprotein DARPP-32 that is activated by the D1 dopamine receptor in limbic brain regions is correlated to this sociosexual investigatory behavior. Neurons immunopositive for phosphorylated DARPP-32 were not only less dense in the nucleus accumbens of males who spent more time with other males, but also more dense in the preoptic area of males who spent more time with females. The use of phosphorylated DARPP-32 as marker for sociosexual exposure is novel in a lizard species. Taken together, in support of previous studies, these results show that differences in embryonic environment stem primarily from incubation temperature, can explain behavioral differences in adulthood in the absence of hormones, and, in concert with hormonal manipulation, can influence neuronal marker sensitivity to sociosexual exposure. / text

Ectotherms (including marine turtles) being especially sensitive to climate, are at risk to the accelerated rate of human-driven climate change. This study addresses two concerns associated with marine turtles and climate change – the relationship between the timing of marine turtle nesting and sea surface temperature; and the concern over the feminization of marine turtle populations due to rising sand temperatures. Previous studies of loggerhead sea turtles (Caretta caretta) and green sea turtles (Chelonia mydas) have documented the relationship between sea surface temperatures and nesting phenology. Earlier nesting behaviors in both species have been associated with warmer sea surface temperatures. Also, sex determination for marine turtles is temperature-dependent. Due to current sand temperatures, it is estimated that loggerhead (Caretta caretta) nests along the Atlantic coast of Florida already produce over 89% female hatchlings. Using shade to reduce nest temperature and increase the proportion of male hatchlings is one option for mitigating the impacts of climate change on marine turtle sex ratios. In this study, a 21- year (1988-2008) dataset of hawksbill sea turtle (Eretmochelys imbricata) nesting at Buck Island Reef National Monument, St. Croix, U. S. Virgin Islands was analyzed in a similar manner to previous studies. It was found that warmer sea surface temperatures were associated with longer nesting seasons and later median nesting dates. Additionally, a preliminary sand shading study was conducted in the first field season (2011) with a subsequent loggerhead nest shading study in the following field season (2012). Although hatching success was not significantly impacted, temperatures were significantly reduced in the majority of shaded nests. This practice may not be immediately applicable as a means of managing sex ratios, but it could be used to reverse the temperature effects of nest relocation

<p>Sex determination is a critical biological process for all sexually reproducing animals. Despite its significance, evolution has provided a vast array of mechanisms by which sexual phenotype is determined and elaborated even within amniote vertebrates. The most prevalent systems of sex determination in this clade are genetic and temperature dependent sex determination. These two systems are sometimes consistent within large groups of species, such as the mammals who nearly ubiquitously utilize XY genetic sex determination, or they can be much more mixed as in reptiles that use genetic or temperature dependent systems and even both simultaneously. The turtles are a particularly diverse group in the way they determine sex with multiple different genetic and temperature based systems having been described. We investigated the nature of the temperature based sex determination system in Trachemys scripta elegans to ascertain whether it behaved as a purely temperature based system or if some other global source of sex determining information might be apparent within thermal regions insufficient to fully induce male or female development. These experiments found that sex determination in this species is much more complex and early acting than previously thought and that each gonad within an individual has the same sexual fate established enough that it can persist even without further communication between. We established a best practice for the assembly and annotation of de novo whole transcriptomes from T. scripta RNA-seq and utilized the technique to quantify the gene regulatory events that occur across the thermal sensitive period.</p><p>Evidence is entirely lacking on the resolution of TSD when eggs are incubated at the pivitol temperature in which equal numbers or males and females are produces. We have produced a timecourse data set that allowed for the elucidation of the gene expression events that occur at both the MPT and FPT over the course of the thermal sensitive period. Our data suggests that early establishment of a male or female fate is possible when temperature is sufficiently strong enough as at MPT and FPT. We see a strong pattern of mutually antagonistic gene expression patterns emerging early and expanding over time through the end of the period of gonad plasticity. In addition, we have identified a strong pattern of differential expression in the early embryo at stages prior to the formation of the gonad. Even without the known systemic signaling attributed to sex hormones emanating from the gonad, the early embryo has a clear male and female gene expression pattern. We discuss how this early potential masculinization or feminization of the embryo may indicate that the influence of temperature may extend beyond the determination of gonadal sex or even metabolic adjustments and how this challenges the well-defined paradigm in which gonadal sex determines peripheral sexual characteristics.</p> / Dissertation

Components of the molecular pathway underlying gonadogenesis in organisms with temperature-dependent sex determination (TSD) have been retained from genetic sex determination. Furthermore, although much of this network has been conserved, new functions for these genes have evolved in this different mode of sex determination. We find that the transcription factors Sox9 and Dmrt1 and the hormone Mis are involved in the formation of a testis and/or the repression of an ovary at a male-producing temperature. While Mis expression may be maintained by Sox9, the initial upregulation of Mis in the developing testis is most likely modulated by some other upstream factor. Dmrt1 appears to play an upstream role in testis sex determination. We provide evidence that the transcription factor Dax1 and the signaling molecule Wnt4, cloned for the first time in an organism with TSD, play roles in gonadogenesis in both sexes. Finally, we show that the transcription factor FoxL2 and the signaling molecule Rspo1 are involved in the formation of an ovary and/or the repression of a testis at a female-producing temperature. In the first investigation of Rspo1 in any organism exhibiting TSD, we demonstrate it is involved upstream in ovarian sex determination. Complementary to descriptive studies, we optimize a whole organ culture system in which gonad explants develop in vitro for up to three weeks. We show that expression of the sex-determining network in isolated gonads mimics in ovo patterns, revealing an endogenous temperature-sensing mechanism that does not require other embryonic tissues. Ectopic expression of Sox9 reveals a possible positive feedback regulation of Dmrt1. The use of this culture system opens the door to functional manipulation of the gonad at the molecular level and is suitable for a myriad of future studies. This work makes strides in elucidating the molecular network underlying gonadogenesis in an organism exhibiting TSD, and invites investigation of the evolution of gene function. The data lend insight into the changing roles of molecules in sex determination across diverse taxa, and into the evolution of developmental pathways in general. / text

In aquatic turtles, females select nest sites that have a high degree of solar exposure, and exploit recently tilled agricultural fields for nesting, presumably because of increased solar exposure and/or easier nest excavation, and the importance of incubation temperature on survival and offspring phenotype. These same disturbed sites are often contaminated by pollutants and turtles can incorporate high levels of pollutants into their eggs which negatively impact hatch success. For my M.S. research, I investigated turtle nest site selection in a system dominated by agricultural and industrial land use, the impact of crop growth on the thermal and hydric dynamics of turtle nests, and I used paired field and laboratory experiments to examine the individual and interactive impacts of agricultural land use and Hg contamination on hatch success and offspring phenotype in Chelydra serpentina. Of the 150 turtle nests found during this research, 84% were located in human-disturbed soils. Nest site characteristics were similar among nests found in Hg contaminated and reference areas. Agriculture and control nests did not differ in temperature at the time of nesting, but temperatures diverged as crops grew, with temperatures in nests in agricultural fields averaging 2.5 °C lower than control nests over the course of incubation. Similarly, despite no initial difference, nest moisture levels diverged throughout incubation and moisture averaged 107 kPa lower in agricultural than control soils throughout incubation. In my field and laboratory experiments, I found that in comparison to turtles from control incubation conditions (i.e., warmer), turtles incubated under agricultural thermal regimens (i.e., colder) took longer to hatch, hatched at smaller structural body sizes, lost more mass after hatching, had lower post-hatching structural growth rates, and were more likely to be male. Additionally, thermal conditions associated with agricultural land use interacted with high levels of mercury to impact hatching success and offspring sex ratios. My thesis research provides one of the first documentations of negative interactive effects of mercury pollution and habitat quality on early vertebrate development and highlights the importance of examining the combined influence of multiple global changes on biological systems. / Master of Science

Gaining a good understanding of marine turtle mating systems is fundamental for their effective conservation, yet there are distinct gaps in our knowledge of their breeding ecology and life history, owing largely to the difficulty in observing these highly mobile animals at sea. Whilst multiple mating by females, or polyandry, has been documented in all marine turtle species, the fitness consequences of this behaviour have not been fully investigated. Furthermore, male mating patterns, operational sex ratios and the number of males contributing to breeding populations are poorly understood, impeding accurate assessments of population viability. In this thesis, I use molecular-based parentage analysis to study, in detail, the genetic mating system of two green turtle (Chelonia mydas) populations. In the focal population in northern Cyprus, I show that, despite exhibiting a strongly female-biased hatchling sex ratio and contrary to our expectations, there are at least 1.3 breeding males to every nesting female. I go on to assess the breeding frequency of male turtles in the population and determine that males do not breed annually at this site, demonstrating that the observed relatively equal sex ratio of breeders is not the result of a few males mating every year, but that the number of breeding males in the population is greater than expected. I show that 24% of nesting females in the population produce clutches with multiple paternity, but do not detect any fitness benefits to polyandrous females, and discuss the potential role of sexual conflict in influencing female mating decisions. Finally, I reveal a high frequency of multiple paternity in green turtle clutches on Ascension Island, one of the largest green turtle rookeries in the world, and discuss possible causes of variation in the level of polyandry among marine turtle populations. The results presented here shed new light on aspects of marine turtle mating systems that are challenging to study, and illustrate the value of molecular data, not only in describing mating patterns, but in elucidating aspects of life history and behaviour that would otherwise be very difficult to ascertain.

Much of what we know about temperature-dependent sex determination (TSD) in reptiles stems from constant temperature incubation studies in the laboratory. In recent years, as TSD studies moved into the field it became evident that TSD was much more complex than previously thought. The present study attempted to reveal the complexity of TSD, as it relates to other features of the species' biology and physical characteristics tractable only in the field, such as fluctuations in incubation temperature and reproductive life history. To this end I studied the ecology of the turtle Carettochelys insculpta, a TSD species inhabiting the wet-dry tropics of northern Australia from 1996 to 1998. I tested hypotheses associated with movements, activity, behaviour, reproduction, nest site choice, nest temperatures, embryonic survival, embryonic aestivation, hatch-ling sex ratios, and emergence in the species. Each of these was also considered in the context of the influence of the wet-dry tropics. Compared to other turtles inhabiting lotic habitats, C. insculpta occupied considerably larger home ranges, covering up to 10 km of river. Of previously published factors influencing home range size, low productivity of the (micro) habitat may best explain the extensive home ranges in C. insculpta. Patchiness and low nutrient value of the chief food (aquatic vegetation) of C. insculpta may force turtles to cover large expanses of river to acquire sufficient energy for growth and reproduction. Females were more active, moved farther, and occupied larger home ranges than males. Home ranges of females comprised 1-4 activity centres, many of which were associated with thermal springs. I suggest that females may exhibit increased activity and movements relative to males because of sexual inequality in parental investment, where food is particularly limiting (e.g., in species with biennial reproduction). Biennial reproduction in the population allowed the examination of the influence of reproductive condition on home range size, movements, and activity. Reproductive condition did not influence home range or activity, but gravid turtles moved father between successive sightings than non-gravid females. Individual data corroborate these findings, with females moving farther between successive sightings while gravid compared to while spent. Contrary to previous reports, turtles did not appear to move into estuarine areas or lowland flood plains during the wet season, but moved into the riparian forest and possibly into wetlands adjacent to the main channel in the vicinity of their dry season home ranges. During the study I documented the turtles' use of small, localized thermal springs discharging from the river bottom. Dataloggers attached to the carapace to monitor ambient water temperatures recorded the frequency and duration of thermal spring use by individuals. Turtles used the thermal springs frequently during the winter (4-6 months) when river temperatures were lower than that of the thermal springs (8 = 29 � 0.52� C). Turtles often utilized thermal springs for several consecutive hours, leaving the springs only to surface for air. Thermal springs may be derived from ground water (which maintains a temperature equivalent to the annual mean air temperature), rather than from a specific geothermal heat source. Nine of 19 radio-telemetered adult females were seen to use thermal springs, of which seven were gravid and two non-gravid. Thus, gravid turtles may seek thermal springs more than non-gravid turtles. Frequency, duration, and timing of usage collectively suggest active thermoregulation as the primary function of thermal spring use. Utilization of thermal springs probably permits turtles to be more active in cooler months, which may enhance growth rates and accumulation of energy for reproduction. Onset of nesting along river stretches with thermal springs preceded nesting in a stretch not known to have thermal springs by 24 days. Thus, I speculate that by warming themselves on thermal springs in the months prior to nesting, turtles may have accelerated follicular development and nested earlier. Female C. insculpta matured at ca. 6 kg body mass (38.0 cm carapace length, 30.5 cm plastron length). Turtles produced egg sizes and clutch sizes similar to that of other turtle species of similar size. Turtles reproduced every second year, but produced two clutches in each breeding year, ca. 40 days apart. Thus, it appeared that females were energy limited, possibly due to the low available energy content of the dry season diet (aquatic vegetation). Life history theory predicts that if some costly behaviour is associated with reproduction, skipping years could reduce that cost and allow savings to be directed into future reproduction. The present study revealed no obvious accessory behaviour in the population. Within years, clutch mass did not differ between early (first) and late (second) clutches. However, earlier clutches tended to have more and smaller eggs per clutch but than later clutches, a new finding for turtles that has been demonstrated in lizards and other animals. Because the study spanned both years with 'big' and 'small' wet seasons, I was able to examine how the magnitude of the wet season influenced reproductive characteristics. Following big wet seasons turtles produced larger, heavier, and more eggs per clutch than they did after small wet seasons. Relationships among body size, egg size, and clutch size were evident after two big wet seasons but not apparent after two small wet seasons. Collectively, annual variation in reproductive characteristics and current life history theory suggest that a big wet season is a plentiful time for the turtles. I investigated beach selection of nesting pig-nosed turtles (Carettochelys insculpta) along a 63 km stretch of river in 1997 and 1998. I used three classes of beaches to examine beach choice: beaches with nests, beaches with only crawls, andbeaches without nests or crawls. Across these beach types I compared aspect, solar exposure, temperature, substrate moisture, height, water depth at approach, and the height of cohesive sand. I located 82 nesting beaches with 221 nests, and identified 171 potential nesting beaches based on previously published criteria. Beaches with nests had a greater substrate moisture content and corresponding higher cohesive sand line (hereafter CSL) than beaches without nests. Beaches with nests also had a higher CSL than beaches with only crawls. Apparently, turtles could not excavate a nest chamber above the CSL due to loose substrate consistency causing sand to fall in on itself. Turtles could only nest at low elevations below the CSL on beaches with lower substrate moisture. Turtles apparently avoided nesting on these beaches due to the higher probability of nest flooding, as corroborated by a concurrent study. Beach temperatures increased with a seasonal increase in air temperatures, and were influenced by aspect and total angle of solar exposure. Temperatures did not differ among beaches with nests, beaches with only crawls, and beaches without crawls or nests. Therefore, there was no indication that turtles were manipulating offspring sex through choice of nesting beach. However, turtles may be manipulating sex by nesting in areas with particular thermal characteristics within beaches. Two related aspects of hatchling emergence were studied. Using emergence phenology data, nest temperatures, historical weather data, and a developmental model, I tested the hypothesis that delayed hatching occurred in C. insculpta, and that such a delay would allow hatchlings to time their emergence to match the onset of the wet season. Hatchling C. insculpta emerged, on average, 17 days later than dates predicted from a developmental model. Combined with observations of hatchlings remaining in eggs until emergence, these results confirmed delayed hatching in nature. This delay was synchronized with initial river rises associated with the onsetof wet season rains, and is consistent with published criteria for embryonic aestivation. On a diel scale, I generated predictions of two potentially competing models for nocturnal emergence in hatchling turtles, based on the knowledge that air temperatures decrease with season during the emergence period. A test of those predictions for C. insculpta produced ambiguous results. However, further analysis indicated that C. insculpta, and probably other nocturnally emerging turtle species, respond to a decline in diel temperature rather than an absolute temperature. The former would ensure nocturnal emergence, while the latter is experienced during the day as well as at night. Nocturnal emergence may be associated with nesting in open microhabitats. The 'decision' of when and where to nest can influence both offspring survival and hatchling sex ratios in animals with temperature-dependent sex determination (TSD). Knowledge of how these maternal attributes influence the incubation environment is an important first step in hypothesizing why TSD evolved in a particular species. 1 studied the influence of nest site choice and timing of nesting on embryonic survival and hatchling sex ratios. Predation and flooding were the major sources of embryonic mortality. Embryonic survival was influenced by both lay date and nest site choice: In one year when nesting began later, nests laid later and at lower elevations were destroyed by early wet season river rises. In other years early nesting precluded flood mortality. However, turtles did not nest at the highest available elevations. I hypothesized that turtles were unable to nest at higher elevations because the sand was dry and not cohesive. A field experiment demonstrated that turtles were constrained to nest at lower elevations where they could construct a nest chamber. A mathematical model predicting hatchling sex from fluctuating temperatures was applied to temperature data from 102 natural nests. Resultsconfirmed a type la pattern of TSD, whereby males are produced from cooler temperatures and females from warmer temperatures. The principal determinant of hatchling sex was lay date. Clutches laid earlier in the season produced mainly males, while later clutches yielded mostly females, due to seasonal ramping of air and sand temperatures. However, nest site choice also exerted an influence on hatchling sex. Female-producing clutches were deposited at higher elevations than male-producing clutches. The onset of nesting was not influenced by water temperatures, but may have been related to the magnitude of the previous wet season(s). Turtles nested earlier after two 'big' wet seasons and later following two 'small' wet seasons. This pattern indicates that the wet season is a plentiful time for the turtles. Adaptive 'differential fitness' models for the evolution of TSD have recently been reviewed and clarified. The differential fitness model that best fits C. insculpta is the 'timematching' model, whereby one sex benefits more than the other from early hatching. Male C. insculpta hatched 2-3 weeks earlier then females, on average. Benefit to early hatching males and, therefore, the ultimate selective mechanism (e.g., growth, time to mature) is unknown. Obtaining such data will likely prove difficult in such a long-lived species. A recent adaptive explanation for the evolution and maintenance of temperaturedependent sex determination (TSD) in reptiles rests upon the assumption that mothers can predict or manipulate offspring sex. I postulated that four physiological and behavioural criteria must be met in order for this assumption to be valid: (1) a strong correlation must exist between substrate temperatures during nest site choice and nest temperatures during the period of development when sex is determined in the egg (thermosensitive period = TSP). (2) Assuming that (1) is possible, mothers would need to be capable of correcting for temporal factors obscuring the predictable thermalcharacteristics of nest sites. This could be accomplished in two ways. By contracting nesting times mothers could assess the relative temperatures of alternate nest sites with some accuracy. A protracted distribution of nesting times could greatly reduce a mother's ability to distinguish between, for example, a cooler nest site at a warmer time and a warmer nest site at a cooler time. Alternatively, mothers would need to be able to incorporate temporal changes in nest site temperatures. (3) Sufficient variation in thermal profiles among nest sites, relative to the breadth of temperatures producing both sexes (pivotal temperatures), would be necessary. For example, if most nests produced both sexes, then depth of the eggs would be the deciding factor determining sex, leaving little opportunity for nest site choice to produce one sex or the other. (4) Mothers would need access to nest sites spanning a range of thermal profiles in order to produce either offspring sex. To this end, home range size relative to the number and location of nesting beaches should be important. I tested these four predictions in Carettochelys insculpta, a beach nesting turtle with TSD, using three years of field data on nest site choice, nesting times, thermal characteristics of nests, hatchling sex ratios, and movements of nesting turtles. A strong positive correlation existed between assessable substrate temperatures at nest site choice and mean daily TSP temperatures in all three years. However, the proportion of explained variation was highly variable among years, and low in 1998. Accordingly, the proportion of nests in which substrate temperatures at nest site choice predicted offspring sex correctly was low in 1998 (48- 62 %, depending on treatment of the data). Nesting times were normally distributed, and combined with diel changes in nest site temperatures greatly reduce a turtle's ability to distinguish between sites that would produce different sexes. Considerable among-clutch variation in thermal profiles to produce variable sex ratios existed, agreeing with other studies on turtles. Radiotelemetry indicated that home rangesencompassed several nesting beaches with differing thermal profiles, indicating scope for producing the desired sex. However, the seasonal increase in air temperatures resulted in an overriding effect of mostly males being produced in early (first) clutches and mainly females being produced in late (second) clutches. Collectively, the results suggest that C. insculpta mothers would find it difficult to predict, and therefore, manipulate hatchling sex, supporting the conventional notion that TSD mothers have little or no control over offspring sex.

Captive husbandry programs in zoos have documented nesting behavior and have successfully hatched Manouria emys emys, but data on sex determining mechanisms and sex ratios are absent. A total of 30 M. e. emys eggs were artificially incubated at five different temperatures in constant humidity. Mean incubator temperatures were 24.99°C, 25.06°C, 27.18°C, 28.00°C, and 30.79°C. Incubation duration ranged from 60 days to 92 days, and hatching success was 50%. Sex determined by histology and laparoscopy resulted in male differentiation at low temperatures (24.99°C, 27.18°C) and female differentiation at high temperatures (30.79°C). Pivotal temperature was estimated to be 29.29°C. The following investigation into temperature-dependent sex determination (TSD), including its presence or absence, pattern, and pivotal temperature, has implications for studies of adaptive significance of reproductive behaviors and of chelonian phylogenetic history. Additionally, the proposed study can provide foundations for conservation management decisions, and for captive breeding programs.