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1. Over half of all crocodylians are threatened with extinction, with many species considered to be functionally extinct throughout their historical ranges. How such losses impact crocodylian functional diversity (FD) and its conservation is poorly understood, but can be investigated using measurable traits. Where such information is unavailable, phylogenetic diversity (PD) has been proposed as an effective strategy to capture FD, but this assumption is not well tested. 2. We constructed a global trait database for 28 extant crocodylians, and quantified their FD, distinctiveness and groupings. Functionally distinctive crocodylians include the most and least evolutionarily distinct species, likely because unusual trait values and combinations can be produced by both evolutionary isolation and evolutionarily recent adaptive radiation. We identified four functional groups of crocodylians, within which species have similar ecological roles. Highly distinctive species, such as the gharial, fit less well into functional groups. 3. We combined functional distinctiveness (FUD) and threat ranking scores to prioritise species for conservation. The metric is termed Ecologically Distinct and Globally Endangered (EcoDGE), and we tested how well it operates to preserve crocodylian FD among extant Crocodylia. Under current extinction scenarios, 32%–38% of crocodylian FD might be lost within 100 years. Losses increase to 48% if all threatened species go extinct, with Asia identified as a hotspot of threatened FD in crocodylians. Highly threatened distinctive species lead this extinction scenario, exacerbated by clusters of species sharing traits linked to high extinction risk. These traits include habitat specialism and lower reproductive investment; in contrast, species that tolerate climate extremes are more resilient. 4. Prioritising at-risk species based on FUD and threat status appears to be an effective way to conserve present-day crocodylian diversity. Conservation prioritisation based on the EcoDGE metric performed well to minimise projected losses in FD. Among extant crocodylians, FD losses are projected when high diversity overlaps with high extrinsic threats. 5. We then examined the utility of PD-based measures as surrogates for FD conservation, to better understand the advantages and drawbacks of such substitutions. A PD-based measure such as the Evolutionarily Distinct and Globally Endangered metric provided an effective tool to capture FD in our tests.

The gharial (Gavialis gangeticus Gmelin) is a fish-eating specialist crocodylian, endemic to south Asia, and critically endangered in its few remaining wild localities. A secondary gharial population resides in riverine-reservoir habitat adjacent to the Nepal border, within the Katerniaghat Wildlife Sanctuary (KWS), and nests along a 10 km riverbank of the Girwa River. A natural channel shift in the mainstream Karnali River (upstream in Nepal) has reduced seasonal flow in the Girwa stretch where gharials nest, coincident with a gradual loss of nest sites, which in turn was related to an overall shift to woody vegetation at these sites. To understand how these changes in riparian vegetation on riverbanks were related to gharial nesting, we sampled vegetation at these sites from 2017 to 2019, and derived an Enhanced Vegetation Index (EVI) from LANDSAT 8 satellite data to quantify riverside vegetation from 1988 through 2019. We found that sampled sites transitioned to woody cover, the number of nesting sites declined, and the number of nests were reduced by > 40%. At these sites, after the channel shift, woody vegetation replaced open sites that predominated prior to the channel shift. Our findings indicate that the lack of open riverbanks and the increase in woody vegetation at potential nesting sites threatens the reproductive success of the KWS gharial population. This population persists today in a regulated river ecosystem, and nests in an altered riparian habitat which appears to be increasingly unsuitable for the continued successful recruitment of breeding adults. This second-ranking, critically endangered remnant population may have incurred an "extinction debt" by living in a reservoir that will lead to its eventual extirpation.

The sex of American Alligator (Alligator mississippiensis) hatchlings is deter- mined by the egg temperature during the middle third of the 9-12 week incubation period. As a consequence, predictable sex ratios are possible for clutches incubated in constant temperatures in the laboratory, but naturally occurring sex ratios of American Alligator hatchlings from wild nests exposed to fluctuating temperatures are not well documented. Over a 5-year period (1995-1999), we determined the sex of American Alligator hatchlings from wild nests left in the field until after sex was irreversibly determined. A total of 6226 hatchlings from 232 naturally incubated wild nests showed a strong female bias (71.9% females, yearly range = 62.3-89.4% females). Most nests (64.2%) produced hatchlings of both sexes. Of the remaining clutches that produced exclusively one sex (83 nests), 78 nests produced all females, and 5 nests produced only male hatchlings. For the 2 years in which nest-cavity temperatures were known, higher temperatures led to production of significantly more male hatchlings (P < 0.001 for both 1997 and 1999). Knowledge of natural sex ratios of hatchlings can aid in the management and harvest of this commercially valuable species, and in understanding sex-ratio bias in American Alligator populations.

Detailed observations on groups of captive saltwater crocodile (Crocodylus porosus) hatchlings revealed sporadic periods of intense agonistic interactions, with 16 highly distinctive behaviours, in the morning (06:00–08:00 h) and evening (17:00–20:00 h) in shallow water. Ontogenetic changes in agonistic behaviour were quantified by examining 18 different groups of hatchlings, six groups each at 1 week, 13 weeks and 40 weeks after hatching. Agonistic interactions between hatchlings at 1 week of age (mean 7.3 ± 0.65/night) were not well-defined and varied in intensity (low, medium, high), number of individuals that were aggressive, and the outcome, while most interactions involved contact (94.5%). There were also clutch specific differences in the frequency of agonistic interactions. At 13 and 40 weeks, a more hierarchal dominance relationship appeared to be established which primarily involved aggression–submission interactions. Agonistic interactions were more frequent (13 weeks 9.7 ± 0.61/night; 40 weeks 22.2 ± 0.61/night) and intense (medium, high), but shorter in duration, in which the subordinate individual fled in response to an approach by a dominant animal that often gave chase but did not make contact. While the full repertoire of behaviour was displayed by hatchlings at 1 week of age, a smaller subset based on dominance status was displayed among 13- and 40-week-old hatchlings. Agonistic behaviour occurs in C. porosus shortly after hatching and is important in establishing and maintaining dominance hierarchies that are characterised by aggression–submission interactions. This type of interaction appears typical for C. porosus both in the wild and in captivity, and may be important in preventing serious injury in a species equipped with formidable armoury. Dispersal by hatchling C. porosus at around 13 weeks of age appears to be driven by a growing intolerance of conspecifics, while territoriality is apparent at an early age. Consequently, agonistic behaviour and social status may be major contributors to the observed differences in growth rates and survival in captivity.

Egg inundation often results in poor hatching success in crocodylians. However, how tolerant eggs are to submergence, and/or how eggshell ultrastructure may affect embryo survival when inundated, are not well understood. In this study, our objective was to determine if embryo survival in Caiman latirostris is affected by eggshell surface roughness, when eggs are submerged under water. Tolerance to inundation was tested early (day 30) versus late (day 60) in development, using eight clutches (four per time treatments), subdivided into four groups: (N = 9 per clutch per treatment; 9 × 4 = 36 eggs per group). ‘Rough’ eggshell represented the natural, unmodified eggshell surface structure. ‘Smooth’ eggshell surface structure was created by mechanically sanding the natural rough surface to remove surface columnar elements and secondary layer features, e.g. irregularities that result in ‘roughness’. When inundated by submerging eggs under water for 10 h at day 30, ‘smooth’ eggshell structure resulted in more than twice as many dead embryos (16 versus 6, smooth versus rough; N = 36), and fewer than half as many healthy embryos (6 versus 13, smooth versus rough, respectively; N = 36). By contrast, at day 60, inundation resulted in very low hatching success, regardless of eggshell surface structure. Only two hatchlings survived the inundation, notably in the untreated group with intact, rough eggshells. Inundation produced a high rate of malformations (58% at day 30), but did not affect hatchling size. Our results indicate that eggshell roughness enhances embryo survival when eggs are inundated early in development, but not late in development. Apparently, the natural surface ‘roughness’ entraps air bubbles at the eggshell surface during inundation, thereby facilitating gas exchange through the eggshell even when the egg is submerged under water.

Three month old hatchling Crocodylus porosus with data loggers in their stomachs were placed in thermal gradients, in isolation ( N =16) and in groups of 4 ( N =8 groups; 32 individuals). Mean T b and variation in T b (SD) was not different whether individual crocodiles in isolation were fasted or fed, or if individuals were housed in isolation ( I ) or in groups ( G ). However, individuals in isolation ( N =16) maintained slightly lower T b s than those in groups ( N =32) during the early morning (06:00–11:00 h). The overall mean T b recorded for fasted individuals in the isolated and group treatments ( N =48) was 30.9±2.3 °C SD, with 50% of T b s ( T set ) between 29.4 °C and 32.6 °C, and a voluntary maximum and minimum of 37.6 °C and 23.2 °C respectively. During the day (11:00–17:00 h), individuals in isolation and in groups selected the warmer parts of the gradient on land, where they moved little. Outside of this quiescent period (QP), activity levels were much higher and they used the water more. There was a strong diurnal cycle for fasted individuals in isolation and in groups, with T b during the QP (31.9±2.09 °C; N =48) significantly higher than during the non-quiescent period (NQP: 30.6±2.31 °C). Thermal variation (SD) in T b was relatively stable throughout the day, with the highest variation at around dusk and early evening (18:00–20:00 h), which coincided with a period of highest activity. The diurnal activity cycle appears innate, and may reflect the need to engage in feeding activity at the water's edge in the early evening, despite ambient temperatures being cooler, with reduced activity and basking during the day. If so, preferred T b may be more accurately defined as the mean T b during the QP rather than the NQP. Implications for the thermal environment best suited for captive C. porosus hatchlings are discussed.

Sex ratios of the American snapping turtleChelydraserpentina are female-biased at cool temperatures, male-biased at moderate temperatures, and only females are produced at warm temperatures. The laboratory incubation of eggs at several constant temperatures yields patterns of skewed sex ratios. In this study, incubated eggs represented regional samples of C. serpentina from six different latitudes. The sample from the highest latitude yielded malebiased sex ratios across the broadest range of cool temperatures. This male-biased zone and those of the other five samples collectively contributed to a nested arrangement that is approximately symmetrical around a temperature of 24.5 ◦ C. Two indices of temperature-dependent sex determination (TSD) patterns (MF pivotal temperature, width of the male-biased zone) were significantly associated with latitude and with published data on several climatic variables (e.g. sunshine and rainfall). At high latitudes, females nest in exposed locations, locally the warmest places. At low latitudes females place their nests in shaded locations. Such nest site selection renders high latitude nests warmer than low latitude nests, even if only intermittently. Given that constant conditions show faster embryogenesis in high vs low latitude eggs, exposed nest sites seem to be an additional adaptation to hasten embryogenesis in high latitude nests. At low latitudes, shading prevents nests from overheating. We propose that a trade-off between evolution, (1) to balance sex ratio and (2) to maintain male-biased sex ratios in a temperature range that favours male fitness, leads to the observed arrangement of concentric TSD patterns centred around male production at 24.5 ◦ C.

Many oviparous reptiles exhibit temperature-dependent sex determination (TSD); i.e., the temperature at which the egg is incubated determines the sex of the offspring. In TSD reptiles, yolk steroids not only may influence sex determination, but also may mediate hormonal effects on subsequent growth and behavior, as in some avian species. We investigated changes in the levels of estradiol (E(2)) and testosterone (T) during development in yolks of snapping turtle eggs, examined how incubation temperature affects hormone levels, and determined how hormones in turtle eggs are influenced by individual females (=clutch effects). Results indicate significant decreases in both hormones (50% decline) by the end of the sex-determining period, when two-thirds of the development is complete. The declines in both E(2) and T were significantly affected by incubation temperature, but in different ways. Eggs incubated at female-producing temperatures maintained high levels, those incubated at male-producing temperatures had low E(2) values, and eggs incubated at pivotal temperatures had intermediate levels of E(2). At all three temperatures, T values underwent significant but approximately equal declines, except during the developmental stages just after the sex-determining period, when T levels decreased more at the male-producing temperature than at either of the other two temperatures. Initially, there were significant clutch effects in both hormones, but such differences, attributable to individual females, were maintained only for E(2) later in development. Here we report for the first time that incubation temperature significantly affects the hormonal environment of the developing embryo of a turtle with temperature-dependent sex determination. Based on this and related findings, we propose that yolk sex steroids influence sexual differentiation in these TSD species and play a role in sex determination at pivotal temperatures.

Although yolk steroids appear to play important roles in the development, growth, and behavior of some birds, their effects in oviparous reptiles are largely unknown. These investigations were initiated to determine initial levels of steroid hormones in the yolks of eggs from two turtle species. Clutches of snapping turtle (Chelydra serpentina) and of painted turtle (Chrysemys picta) eggs were collected and individual egg yolks were analyzed for estradiol-17beta (E(2)) and testosterone (T) using specific RIAs. E(2) and T levels differed significantly between species, the mean E(2) value in snapping turtles was 2.78+/-0.095 (ng/g) compared to 0.89+/-0.064 (ng/g) for painted turtles, and the mean value for T in snapping turtle yolks was 2.56+/-0.098 (ng/g) compared to 0.68+/-0.045 (ng/g) for painted turtles. In addition, E(2) levels were greater than T levels in both species. Within each species, there were significant differences among clutches from different females. E(2) levels in the snapping turtle yolks varied from a clutch mean of 1.38 to 4.55 ng/g and in painted turtles, the clutch means for E(2) varied from 0.34 to 1.34 ng/g. T levels demonstrated similar phenomena within species, with levels in snapping turtles varying from a clutch mean of 0.68 to 4.71 ng/g. Painted turtle levels of T varied from a clutch mean of 0.22 to 0.72 ng/g. There were also significant differences in the E(2)/T ratio, however, E(2)/T ratios did not differ between species. Painted turtle follicles of different sizes showed significant differences in levels of both E(2) and T, and these differences may reflect differing deposition patterns of these steroids in the egg yolk of this turtle during vitellogenesis. The differences in E(2) and T concentration reported here could have important implications for development, growth, and behavior in oviparous reptilian species.

Egg inundation often results in poor hatching success in crocodylians. However, how tolerant eggs are to submergence, and/or how eggshell ultrastructure may affect embryo survival when inundated, are not well understood. In this study, our objective was to determine if embryo survival in Caiman latirostris is affected by eggshell surface roughness, when eggs are submerged under water. Tolerance to inundation was tested early (day 30) versus late (day 60) in development, using eight clutches (four per time treatments), subdivided into four groups: (N = 9 per clutch per treatment; 9 × 4 = 36 eggs per group). ‘Rough’ eggshell represented the natural, unmodified eggshell surface structure. ‘Smooth’ eggshell surface structure was created by mechanically sanding the natural rough surface to remove surface columnar elements and secondary layer features, e.g. irregularities that result in ‘roughness’. When inundated by submerging eggs under water for 10 h at day 30, ‘smooth’ eggshell structure resulted in more than twice as many dead embryos (16 versus 6, smooth versus rough; N = 36), and fewer than half as many healthy embryos (6 versus 13, smooth versus rough, respectively; N = 36). By contrast, at day 60, inundation resulted in very low hatching success, regardless of eggshell surface structure. Only two hatchlings survived the inundation, notably in the untreated group with intact, rough eggshells. Inundation produced a high rate of malformations (58% at day 30), but did not affect hatchling size. Our results indicate that eggshell roughness enhances embryo survival when eggs are inundated early in development, but not late in development. Apparently, the natural surface ‘roughness’ entraps air bubbles at the eggshell surface during inundation, thereby facilitating gas exchange through the eggshell even when the egg is submerged under water. Fil: Cedillo Leal, César. Universidad Autónoma de Tamaulipas; México Fil: Simoncini, Melina Soledad. Provincia de Entre Ríos. Centro de Investigaciones Científicas y Transferencia de Tecnología a la Producción. Universidad Autónoma de Entre Ríos. Centro de Investigaciones Científicas y Transferencia de Tecnología a la Producción. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Centro de Investigaciones Científicas y Transferencia de Tecnología a la Producción; Argentina. Universidad Nacional del Litoral. Facultad de Humanidades y Ciencias; Argentina Fil: Leiva, Pamela Maria de Lujan. Provincia de Entre Ríos. Centro de Investigaciones Científicas y Transferencia de Tecnología a la Producción. Universidad Autónoma de Entre Ríos. Centro de Investigaciones Científicas y Transferencia de Tecnología a la Producción. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Centro de Investigaciones Científicas y Transferencia de Tecnología a la Producción; Argentina. Universidad Nacional del Litoral. Facultad de Humanidades y Ciencias; Argentina Fil: Larriera, Alejandro. Universidad Nacional del Litoral. Facultad de Humanidades y Ciencias; Argentina Fil: Lang, Jeffrey W.. University of Minnesota; Estados Unidos Fil: Piña, Carlos Ignacio. Provincia de Entre Ríos. Centro de Investigaciones Científicas y Transferencia de Tecnología a la Producción. Universidad Autónoma de Entre Ríos. Centro de Investigaciones Científicas y Transferencia de Tecnología a la Producción. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Centro de Investigaciones Científicas y Transferencia de Tecnología a la Producción; Argentina. Universidad Nacional del Litoral. Facultad de Humanidades y Ciencias; Argentina

Ambient temperatures during embryonic development determine gonadal sex in many reptiles. The temperature sensitive period for sex determination has been defined by shifting eggs between female- and male-producing temperatures in a few species. This phase spans 20-35% of embryogenesis in most species, which makes it difficult to define the mechanisms that transduce temperature into a signal for ovarian versus testicular development. We present an extensive set of studies that define a brief period when high temperature specifies, and then determines, ovarian fate in a northern population of snapping turtles, Chelydra serpentina. We shifted embryos from male to female temperatures, or vice versa, at various stages of development. Gonads in embryos incubated at female temperatures commit to ovarian fate earlier (by stage 18) than gonads in embryos incubated at male temperatures commit to testicular fate (by stages 19-21). In double shift studies, embryos were incubated at a female temperature, exposed to a male temperature for set times, and shifted back to the original temperature, or vice versa. The time required to induce ovarian development (≤6 days at female temperatures) was much shorter than the time required to induce testicular formation (>20 days at male temperatures). Differentiation of the gonads at the histological level occurred after the sex-determining period. Nevertheless, we found that a change in temperature rapidly (within 24h) influenced expression and splicing of WT1 mRNA: the absolute abundance of WT1 mRNA, the relative abundance of +KTS versus -KTS isoforms, as well as the ratio of +KTS:-KTS isoforms was higher in gonads at a male versus a female temperature. In conclusion, ovarian fate is more readily determined than testicular fate in snapping turtle embryos. The short sex-determining period in this species (6-8% of embryogenesis) will facilitate studies of molecular mechanisms for specification and determination of gonad fate by temperature.

Recent theoretical papers on temperature-dependent sex determination in reptiles are based on the assumption that crocodilian populations, particularly adults, are markedly female biased. While there is evidence that some crocodile populations may conform to this expectation, there is no compelling evidence that American alligator populations are female biased anywhere in the species' range. Previous data had indicated that some populations of juvenile and/or adult alligators were significantly male biased. However, these studies were criticized for sampling errors. Adult males typically occupy a different habitat from that of females, and males frequent areas where they are more likely to be caught. In contrast, in juveniles, both sexes occupy the same habitat. We determined the sex ratios of c. 3000 juvenile alligators collected from 11 sites over 6 years in south Louisiana. Our results indicate a significant sex bias (58% male), but variation was evident among samples. Sex ratios varied by year and site, and the interaction of these factors was significant. At one site in one year, there was a female bias (71%), but in no year was there an overall female bias across sites. From this study and earlier reports, we conclude that there is no evidence at present of female-biased sex ratios in the juvenile and/or adult alligator populations, and consequently, that certain models of temperature-dependent sex determination in reptiles require re-evaluation. Furthermore, our data suggest a pattern of differential mortality of females vs males during the first years of life, a pattern consistent with a key prediction of several differential fitness models for the adaptive significance of temperature-dependent sex determination in reptiles.

We assessed the ability of hatchling painted turtles (Chrysemys picta) from northern Minnesota to resist freezing when exposed to conditions like those occurring in natural hibernacula (natal nests). We placed animals individually into artificial hibernacula constructed in jars of damp, loamy sand and then lowered the temperature to approximately -0.4°C, which was below the equilibrium freezing point for water in the soil (approximately -0.1°C) but above that for body fluids of the turtles (approximately -0.7°C). Next, ice was used to initiate freezing of water in the soil, after which the substratum was allowed to freeze to an equilibrium temperature of -0.4°C. The minimum temperature then was reduced by 1°C/day to either -2.5 or -6.5°C. The minimum was maintained for 9 days in the former treatment and for 5 days in the latter, so that turtles in both treatments were exposed for the same length of time to temperatures below the equilibrium freezing point for their body fluids. Some animals in each treatment were inoculated by ice crystals that penetrated their integument, and these turtles froze and died. However, many other animals resisted inoculation, remained unfrozen, and survived the test of tolerance. We conclude that hatchling painted turtles from northern Minnesota, like those from North Dakota, north-central Nebraska, and northern Illinois, withstand the cold of midwinter by supercooling and not by tolerating freezing.

Embryonic temperature influenced subsequent growth in juvenile snapping turtles, Chelydra serpentina: incubation temperatures of 24 and 26.5°C enhanced growth relative to a temperature of 29°C. Although embryonic temperature normally determines gonadal sex in this species, experimental manipulations revealed that temperature effects on growth were independent of sex. Ambient temperature also affected growth: juvenile turtles grew slowly in a cool (19°C) versus a warm (28°C) environment. In a parallel experiment, turtles from different embryonic temperatures displayed different patterns of temperature choice in response to nutritional status or time of day. We tentatively conclude that embryonic temperature has both direct and indirect (i.e., through temperature choice) effects on growth in snapping turtles.

Unlike birds and mammals, in many reptiles the temperature experienced by a developing embryo determines its gonadal sex. To understand how temperature-dependent sex determination (TSD) evolves, we must first determine the nature of genetic variation for sex ratio. Here, we analyze among-family variation for sex ratio in three TSD species: the American alligator (Alligator mississipiensis), the common snapping turtle (Chelydra serpentina) and the painted turtle (Chrysemys picta). Significant family effects and significant temperature effects were detected in all three species. In addition, family-by-temperature interactions were evident in the alligator and the snapping turtle, but not in the painted turtle. Overall, the among-family variation detected in this study indicates potential for sex-ratio evolution in at least three reptiles with TSD. Consequently, climate change scenarios that are posited on the presumption that sex-ratio evolution in TSD reptiles is genetically constrained may require reevaluation.

The leading explanation of temperature-dependent sex determination (TSD) in reptiles postulates that (1) ovarian differentiation is directed by estrogen and that (2) estrogen is synthesized in the developing gonad following induction of aromatase expression. However, the source of steroid substrate for aromatization has not yet been identified. In addition, sex ratios vary as a function of clutch, but such biases are as yet unexplained. To address these issues, we measured estradiol, testosterone, and androstenedione in yolks of the American alligator (Alligator mississippiensis) before, during, and after the period of gonadal differentiation in this TSD species. Eggs were collected from a wild population in Louisiana and were incubated at male- and female-determining constant temperatures in the lab, as well as at intermediate temperatures that produced both sexes. Steroids were assayed in yolk extracts after celite column chromatography. All three steroids were found to be in the range of nanograms/gram of yolk at stage 16. Androstenedione was the predominant steroid, 2- to 3-fold higher in concentration than estradiol and 15- to 20-fold higher than testosterone. The levels of these steroids declined (5- to 30-fold) between stages 16 and 25, most markedly between stages 21 and 23, regardless of incubation temperature. The chronology of this sharp decline in steroid levels in our study coincides with the timing of gonadal differentiation in this species, between stages 21 to 23 based on previous reports. Estradiol levels in yolks differed by 3-fold in some clutches relative to others, whereas, no clutch differences were apparent for either androstenedione or testosterone. These data demonstrate that alligator yolk contains high concentrations of two steroid substrates utilized for estrogen synthesis, as well as significant quantities of estradiol itself. We hypothesize that estradiol levels in yolk provide a steroid background, variable among and within clutches, on which gonadal development is initiated and proceeds. As a consequence, we suggest that yolk provides an epigenetic maternal contribution that modulates the effect of incubation temperature on hatchling sex.

In many oviparous reptiles, incubation temperature determines hatchling sex. Manipulation of the embryonic hormonal environment can result in sex reversal. We measured circulating estradiol 17-β (E2) and testosterone (T) levels in temperature-determined and sex-reversed snapping turtles (Chelydra serpentina). Eight-month-old turtles have sex steroid profiles that are sex specific and are consistent with gonadal sex. Males had higher levels of T than females, both before and after treatment with follicle stimulating hormone (FSH). In males, T levels increased approximately 11-fold after FSH challenge, whereas in females, T levels remained low. In contrast, levels of E2 after FSH challenge were higher in females than in males, but showed no difference before treatment. Sex-reversed individuals appear to have normal sex steroid profiles of T and E2, both at basal levels and in response to gonadotropin challenge. Our study indicates that the steroidogenic capability of these juvenile turtles was consistent with gonadal morphology and suggests that these sex-reversed individuals have normal gonadal function. © 1996 Wiley-Liss, Inc.

Abstrcact.-We examined a critical component of the Charnov-Bull hypothesis of temperaturedependent sex determination (TSD) by determining the reaction norms of hatchling growth to embryonic incubation temperature in the common snapping turtle, Chelydra serpentina. Hormone manipulations of eggs produced females at male temperatures and vice versa, which thereby permitted same-sex comparisons of hatchling growth across a range of incubation temperatures. In this way, the normally confounded effects of incubation temperature and sex were dissociated experimentally. The resultant hatchlings, including controls and experimentals, exhibited normal gonadal structure and sex steroid profiles. The subsequent growth of hatchlings monitored for 6 mo was strongly affected by embryonic incubation temperature but not by sex. As predicted, growth was enhanced at incubation temperatures that produced males. Clutch effects and interaction effects (clutch by incubation temperature) on growth were significant. In addition, there was a positive genetic covariance among incubation temperatures, but incubation temperature effects varied among clutches. The variation in growth plasticity among clutches was consistent with Charnov-Bull predictions. In this TSD species, incubation temperature is likely to have differential fitness effects on the sexes mediated via differences in growth.

Sexual differentiation of the gonads in Alligator mississippiensis and many other oviparous reptiles is controlled by egg incubation temperature. Estrogens are thought to play a role in this process, and it has been hypothesized that estrogen production is thermosensitive in species with temperature-dependent sex determination (TSD). Using the tritiated water assay, we measured the activity of the critical estrogen-synthesising enzyme, P450 aromatase, in the gonad-adrenal-mesonephric kidney complex (GAM) of alligator embryos incubated at male- and female-producing temperatures. Aromatase activity increased in the GAM of developing embryos incubated at 30° C (100% female-producing) and 34.5° C (predominantly female-producing), while it remained very low throughout development in embryos incubated at the intermediate temperature of 33° C (100% male-producing). However, it is unclear whether enhanced aromatase activity represents the initial signal for ovary differentiation or whether it lies downstream in the female developmental pathway. For embryos incubated at 30° C (female-producing), there was no detectable increase in aromatase activity until developmental stage 24, which is after the temperature-sensitive period for sex determination. This suggests that aromatase may be a downstream component of the ovary-determining cascade. In female alligator hatchlings, most of the aromatase activity was localised in the ovary, activity being low in the adrenal-mesonephros. Aromatase assays carried out at 30° C and at 33° C indicated that, at viable incubation temperatures, aromatase activity is not thermosensitive. This suggests that temperature directly or indirectly influences enzyme synthesis. Radioimmunoassay of estradiol synthesised by the GAM during the aromatase assay confirmed increased enzyme activity during female development but not during male development. Increased aromatase activity and estrogen synthesis during female development were correlated with the timing of ovary differentiation, particularly proliferation of the gonadal cortex. These findings implicate aromatase in temperature-dependent gonadal sex differentiation in alligator embryos, higher enzyme activity being associated with ovary development.

Incubation temperature determines sex in crocodilians and influences other features of hatchlings. We investigated the effects of incubation temperature, clutch, and sex on the dimensions, mass, and energy reserves of hatchling Alligator mississippiensis. Eggs from three clutches were incubated to pipping at constant temperatures of 29°, 31°, 32°, and 33° C; females were produced at 29° and 31° C, mixed sex ratios at 32° C, and males at 33° C Incubation temperature affects hatchling size and yolk utilization in a consistent, but complex, pattern. Maximum yolk-free hatchling mass occurred at 32° C, and minimum mass at 31° C. Similarly, dimensions were maximized at 32° C and minimized at 29° C Residual yolk mass was inversely related to yolk-free hatchling mass. Higher yolk mass occurred at 31° and 33° C; at 29° and 32° C, values were lower. However, fat mass remained constant in all clutches. Strong clutch effects were present in nearly all traits. At 32° C, males were heavier and had less yolk than female...

In certain reptiles with temperature-dependent sex determination (TSD), estrogens act as a signal for female differentiation. Because aromatase produces estrogens from androgens, this enzyme plays a pivotal role in TSD. Whether androgens act as the signal for male differentiation in TSD species in not yet clear. We manipulated the hormonal environment in eggs of the common snapping turtle (Chelydra serpentina) to determine the effects of an estrogen (estradiol 17-beta), an aromatase inhibitor (fadrozole; CGS 16949A), and androgens (testosterone and dihydrotestosterone) on sex determination in this TSD species. Test solutions were applied topically to representative eggs (total tested = 1054 from 27 clutches) and incubated at two male-producing temperatures (24 and 26.5 degrees) and at a predominantly female-producing temperature (29 degrees). In this species, application of an estrogen induced female development at all temperatures tested. In contrast, the aromatase inhibitor had no effect at the male-producing temperatures, but induced male development at the predominantly female-producing temperature. At this temperature, aromatase inhibitor plus testosterone had a similar male-producing effect, but when applied alone, testosterone failed to augment male production. Dihydrotestosterone had a similar effect, in contrast to its reported androgenic effects in other TSD species. In the snapping turtle, male differentiation may not be androgen dependent; rather, it may proceed in the absence of female differentiation. In this species, female development is clearly estrogen-dependent and is altered by aromatase inhibition at female-producing temperatures. Our results not only provide additional evidence that sex steroids mediate gonadal differentiation in TSD species, but also suggest caution with respect to generalizations about the proximal mechanisms of TSD in reptiles.

Genetic fingerprint profiles have been successfully used for establishing biological relationships, in linkage analysis, and in studies of population structure but have not so far been used for ascertaining phylogenetic relationships among related groups of species and genera. This is largely because these profiles are thought to evolve too rapidly to be informative over large time intervals. However, we show here that among the Crocodilia, whose phylogeny is a debated issue, these profiles can provide phylogenetically useful information. By using the probe Bkm-2(8), DNA fingerprints with distinct bands distributed in the size range 0.5-23.0 kb were obtained for individuals of 18 species belonging to seven of the eight genera of crocodilians. These genetic profiles showed individual-, species-, and restriction enzyme-specific patterns. In addition, striking differences were observed in the copy number of Bkm-related sequences in genomes of different crocodilian species. The qualitative data from DNA fingerprint profiles, and quantitative data on copy number variation in Bkm-related sequences, suggest that these genera belong to two distinct groups, one of which includes Alligator, Paleosuchus, and Caiman; the other includes Crocodylus, Osteolaemus, Tomistoma, and Gavialis. A close relationship between Tomistoma and Gavialis is also suggested by these results.

Half of the 22 extant crocodilians show evidence of temperature-dependent sex determination (TSD). We examine evidence for TSD in 11 species by reviewing reports on five and presenting new data for six. The female-male pattern (FM; females at low temperature, males at high temperature) attributed to Alligator mississippiensis and Caiman crocodilus are here revised to be female-male-female (FMF; males at intermediate temperature, females at low and high temperatures). A similar pattern characterizes Crocodylus palustris, C. moreletii, C. siamensis, and Gavialis gangeticus based on new data; published accounts establish a FMF pattern in Crocodylus porosus, C. johnstoni, and C. niloticus. TSD apparently occurs in Paleosuchus trigonatus and Alligator sinensis, but patterns are not yet documented. In the well-studied species, the incubation temperatures for FM transitions are congruent, but MF transition temperatures differ among species. In A. mississippiensis, 100% males are produced over a range of constant incubation temperatures, whereas in C. johnstoni, only low proportions of males are produced at any constant temperature. The thermosensitive period (TSP) for A. mississippiensis occurs during stages 21 to 24 (days 30–45 at intermediate temperatures) and coincides with gonadal differentiation. A similar scenario is suggested in other species. The TSP in A. mississippiensis (and possibly other crocodilians) encompasses the third quarter of development and occurs later than in turtles and a lizard. In A. mississippiensis as in turtles, the duration (cumulative effect) and/or the magnitude (potency effect) of incubation temperatures during the TSP predictably alter sex ratios. TSP chronologies and features which are shared among TSD reptiles suggest common, underlying mechanisms; A. mississippiensis is an appropriate model for further study. In crocodilians, clutch effects are a significant source of variation in TSD response. Hatchling sex ratios previously reported for A. mississippiensis are reconsidered in light of our new data. © 1994 Wiley-Liss, Inc.

Individual-specific DNA fingerprints of crocodilians were obtained by the use of Bkm-2(8) probe. Pedigree analyses of Crocodylus palustris, C. porosus and Caiman crocodilus revealed that the multiple bands (22-23 bands with Aludigest) thus obtained were inherited stably in a Mendelian fashion. Unique fingerprints permitted us to identify individuals, assign parentage, and reconstruct the DNA profile of a missing parent. Average band sharing between unrelated crocodiles was found to be 0.37. Band sharing between animals of known pedigrees increased predictably with relatedness and provided a basis for distinguishing relatives from non-relatives. Similar results obtained in other species/genera, using the same probe, suggest that this approach may be applicable to all species of crocodilians, and could facilitate genetic studies of wild and captive populations.

Blood samples collected in the field for isolating DNA suitable for molecular analysis need special care in their storage and handling. In this article, we describe a simple method for the isolation of good-quality high-molecular-weight DNA that does not require low temperature conditions during collection, storage, and/or transportation of blood samples. This method involves smearing small aliquots of blood onto clean slides and air drying them at room temperature. The slides with blood smears can then be transported or stored at room temperature and still serve as a very good source of high-molecular-weight DNA. Genomic DNA from these samples can be extracted by organic phase separation (phenol-chloroform extraction) after lysis. The DNA thus obtained is of high quality and yields DNA fingerprints qualitatively similar to those prepared from corresponding control DNA isolated from frozen blood samples. Needing minimal facilities at field sites, the method is very convenient for conducting RFLP analysis of wild/field populations for demographic, behavioral, and ecologic studies.

We studied tolerance for cold in hatchling painted turtles (Chrysemys picta) from Lake Metigoshe, Bottineau County, North Dakota, to determine whether neonates in populations near the northern limit of distribution rely on a tolerance for freezing or on a capacity for supercooling to survive their first winter of life. We placed hatchlings individually into artificial hibernacula constructed in jars of damp, loamy sand and then cooled the jars to approximately -0.45 degrees C, which was below the equilibrium freezing point for water held by the sand but above that for body fluids of the neonatal turtles. A piece of ice next was placed on the surface of the sand in each jar to induce freezing of the soil water. After the soil water had frozen to an equilibrium, the temperature in the jars was lowered by 1 degrees C/d to minima averaging -2.5 degrees C, -4.5 degrees C, -6.5 degrees C, and -10.5 degrees C in different treatments. These temperatures were maintained for varying periods, so that animals in each treatment were exposed to temperatures below the equilibrium freezing point for their body fluids for a total of 11 d. Thirty of 32 hatchlings survived exposure to -2.5 degrees C; 24 of 32 survived at -4.5 degrees C; 14 of 32 withstood -6.5 degrees C; and 7 of 32 tolerated -10.5 degrees C. Freezing exotherms were detected in temperature profiles for turtles that succumbed but not in those for hatchlings that survived. Thus, the ability of hatchlings to withstand subzero temperatures for extended periods apparently requires that they avoid freezing. Although other workers contend that tolerance for freezing is the key to survival over winter by hatchling painted turtles from the region of Lake Metigoshe, our findings indicate that neonates rely primarily on their ability to remain unfrozen and supercooled.

We examined agonistic behaviour in seven species of hatchling and juvenile crocodilians held in small groups (N = 4) under similar laboratory conditions. Agonistic interactions occurred in all seven species, typically involved two individuals, were short in duration (5–15 seconds), and occurred between 1600–2200 h in open water. The nature and extent of agonistic interactions, the behaviours displayed, and the level of conspecific tolerance varied among species. Discrete postures, non-contact and contact movements are described. Three of these were species-specific: push downs by C. johnstoni; inflated tail sweeping by C. novaeguineae; and, side head striking combined with tail wagging by C. porosus. The two long-snouted species (C. johnstoni and G. gangeticus) avoided contact involving the head and often raised the head up out of the way during agonistic interactions. Several behaviours not associated with aggression are also described, including snout rubbing, raising the head up high while at rest, and the use of vocalizations. The two most aggressive species (C. porosus, C. novaeguineae) appeared to form dominance hierarchies, whereas the less aggressive species did not. Interspecific differences in agonistic behaviour may reflect evolutionary divergence associated with morphology, ecology, general life history and responses to interspecific conflict in areas where multiple species have co-existed. Understanding species-specific traits in agonistic behaviour and social tolerance has implications for the controlled raising of different species of hatchlings for conservation, management or production purposes.

We examined agonistic behaviour in hatchling Australian freshwater crocodiles (Crocodylus johnstoni) at 2 weeks, 13 weeks, and 50 weeks after hatching, and between C. johnstoni and saltwater crocodiles (Crocodylus porosus) at 40–50 weeks of age. Among C. johnstoni, agonistic interactions (15–23 s duration) were well established by two weeks old and typically involved two and occasionally three individuals, mostly between 17 : 00 and 24 : 00 hours in open-water areas of enclosures. A range of discrete postures, non-contact and contact movements are described. The head is rarely targeted in contact movements with C. johnstoni because they exhibit a unique ‘head raised high’ posture, and engage in ‘push downs’. In contrast with C. porosus of a similar age, agonistic interactions between C. johnstoni were conducted with relatively low intensity and showed limited ontogenetic change; there was also no evidence of a dominance hierarchy among hatchlings by 50 weeks of age, when the frequency of agonistic interactions was lowest. Agonistic interactions between C. johnstoni and C. porosus at 40–50 weeks of age were mostly low level, with no real exclusion or dominance observed. However, smaller individuals of both species moved slowly out of the way when a larger individual of either species approached. When medium- or high-level interspecific interactions did occur, it was between similar-sized individuals, and each displayed species-specific behaviours that appeared difficult for contestants to interpret: there was no clear winner or loser. The nature of agonistic interactions between the two species suggests that dominance may be governed more strongly by size rather than by species-specific aggressiveness.

Hatchling painted turtles (Chrysemys picta), Blanding's turtles (Emydoidea blandingi), slider turtles (Trachemys scripta), and snapping turtles (Chelydra serpentina) were inoculated with ice at temperatures near the equilibrium freezing point for their body fluids (ca. -0.7 C) and then frozen at -2.0 C. All animals survived freezing for up to 30 h, but mortality among sliders and snapping turtles increased rapidly with longer exposures. Despite the fact that neonates of all four species are able to recover from freezing at a high subzero temperature, the adaptive strategy manifested by animals in the field is to avoid freezing altogether via attributes of morphology (painted turtles), behavior (Blanding's turtles, snapping turtles), or distribution (slider turtles). The discovery of a tolerance for freezing in hatchlings of species having diverse distributions and natural histories raises the possibility that such tolerance is a trait of general occurrence among neonatal turtles and that it is not an adaptation specifically enabling animals to withstand the rigors of winter. Hatchlings of the North American painted turtle (Emydidae: Chrysemys picta) typically spend their first winter inside the shallow, subterranean nest where they completed embryogenesis the preceding summer (Ernst et al., 1994). This behavior commonly causes neonates in northerly populations to be exposed during winter to ice and cold (Breitenbach et al., 1984; Storey et al., 1988; Packard et al., 1989; Costanzo et al., 1995), with temperatures in nests sometimes going below -10 C (Woolverton, 1963; DePari, 1996; Packard, 1997; Packard et al., 1997a). Hatchling painted turtles are small (3-6 g), ectothermic animals with limited heat capacity, so their deep body temperature presumably tracks the temperature in their nest quite closely (Claussen and Zani, 1991). Nevertheless, some turtles commonly survive exposure to such temperatures and emerge from their nest when the ground thaws the following spring (Woolverton, 1963; DePari, 1996; Packard, 1997; Packard et al., 1997a). We have proposed that neonatal painted turtles in northerly populations withstand exposure to ice and cold by remaining unfrozen and supercooled (Packard and Packard, 1995a). According to this theory, the skin of hatchlings is not easily penetrated by growing crystals of ice (Packard and Packard, 1993b), so turtles are not necessarily caused to freeze by inoculation when they make contact with ice in frozen soil (Packard et al., 1997b, 1999). This protection from inoculation enables the animals to exploit an innate capacity for supercooling (Packard and Packard, 1993a, 1995b, 1997; Packard and Janzen, 1996; Costanzo et al., 1998), so that they remain unfrozen over the usual range of subzero temperatures encountered in nests in the field (Packard et al., 1997b, 1999). However, on those occasions when the integument is not up to the challenge and ice crystals manage to penetrate into body compartments from the environment, the animals freeze and die (Packard et al., 1997b, 1999). Thus, turtles that emerge from nests in the spring presumably are ones that remained unfrozen and supercooled for the entire winter. However, a nagging problem with the concept that hatchling painted turtles exploit a capacity for supercooling to survive winters in the field is the indisputable evidence from experiments in the laboratory that neonates often recover from freezing at relatively high subzero temperatures (Storey et al., 1988; Churchill and Storey, 1992a; Rubinsky et al., 1994; Costanzo et al., 1995; Attaway et al., 1998). The problem that these findings create for proponents of an adaptive strategy based on supercooling can be reduced to a simple question: Why do animals tolerate freezing if such a tolerance is not important to their ecology? We believe that we have obtained a partial answer to the aforementioned question. Results from the first of two experiments reported here indicate that a tolerance for limited freezing is common to hatchling turtles generally, that is, that such tolerance probably was acquired from some common ancestor of turtles and is a shared (plesiomorphic) character in contemporary forms. The evidence was gathered by studying tolerance for freezing in hatchlings of four species of North American turtles-painted turtles, Blanding's turtles (Emydoidea blandingi), slider turtles (Trachemys scripta), and snapping This content downloaded from 207.46.13.129 on Sun, 26 Jun 2016 06:51:49 UTC All use subject to http://about.jstor.org/terms FREEZING IN HATCHLING TURTLES turtles (Chelydra serpentina)-that differ in geographic distribution and in the natural history of overwintering neonates. All the hatchlings in this experiment were frozen at -2 C and then held at that temperature for up to 48 h before their condition (i.e., alive or dead) was assessed. We also performed a second experiment, in which hatchling painted turtles were frozen at -2 C for 24 h and then exposed for varying intervals to -2 C, -3 C, or -4 C. Data from this second experiment provide new insights into the possible role of freeze-tolerance in enabling neonates of this species to survive winters in the field. MATERIALS AND METHODS Eggs of painted turtles and snapping turtles were collected in June 1996 from newly constructed nests located on the Valentine National Wildlife Refuge in north-central Nebraska, whereas eggs of Blanding's turtles were collected that same month from fresh nests located along the Mississippi River in central Minnesota. Eggs of sliders, on the other hand, were obtained by injecting gravid females from westcentral Illinois with synthetic oxytocin to induce them to oviposit. The eggs of all species were incubated to hatching on damp vermiculite. Hatchling Blanding's turtles and sliders later were transported to Colorado State University by air express. Average mass for hatchlings was 3.7 g (SD = 0.6 g) for painted turtles; 7.7 g (SD = 0.4 g) for Blanding's turtles; 6.0 g (SD = 1.1 g) for slider turtles; and 7.8 g (SD = 1.1 g) for snapping turtles. The turtles were acclimated to approximately 2 C and held in darkness until they were used to study their tolerance for freezing (Packard and Packard, 1993a). Samples of 8-12 turtles were drawn at random from the pools of available animals. Each hatchling in a sample then was prepared for study by cleaning it thoroughly with a small paint brush, after which the measuring junction of a copper/constantan thermocouple (26 gauge) was affixed to its carapace with epoxy resin. When the resin had hardened, the turtle was dipped in chilled (2 C) tap water to wet its integument-especially the skin in the axillary and inguinal pouches, because this integument in painted turtles may be more susceptible than skin elsewhere on the body to penetration by growing crystals of ice (Packard and Packard, 1995b). Each turtle was placed immediately into a pint-volume canning jar, and the sealed jar was placed in an environmental chamber set at approximately 2 C. After all jars were in the chamber, free ends of the thermocouples were attached via a multiplexor to a Campbell CR-10 datalogger so that temperature on the carapace of the animals could be measured every 30 sec. We next allowed temperature of the turtles to equilibrate with that of the environmental chamber. We then opened the chamber, removed the lid from each jar, placed several pieces of crushed ice against the turtle, and closed the jar. After ice had been added to all the jars, the door to the environmental chamber was closed and the microprocessor controlling the chamber was reset to a nominal temperature of -2 C. Temperature in the chamber declined rapidly, and so too did surface temperature of the turtles (Fig. 1). When temperature reached approximately -0.4 C, ice crystals contacting each turtle caused water on the integument to begin to freeze, as evidenced by a slight decrease in slope of the cooling curve brought about by the release of latent heat of fusion by water changing phase from liquid to solid (Fig. 1). In most cases, the turtle was inoculated shortly thereafter, as evidenced by a pronounced freezing exotherm (i.e., the sudden increase in temperature caused by the release of latent heat of fusion) that lasted for 12-15 h (Fig. 1A, C, D). No freezing exotherm was detected in the temperature profiles for three of the painted turtles (Fig. 1B), so these animals apparently avoided inoculation and remained unfrozen for the duration of their exposure. Timing of each exposure began with the appearance of the freezing exotherm for the turtle itself (Fig. 1A, C, D). Animals in the first experiment comparing hatchlings of all four species were frozen at approximately -2 C for 24-48 h. Some variation occurred in the equilibrium temperatures that actually were measured on the carapace of animals in different jars, owing to the spatial variation in temperature that commonly occurs in environmental chambers (Measures et al., 1973). The average temperature across species and exposures was -2.1 C (range -1.8 C to -2.6 C) and did not vary among species or treatment. Painted turtles in the second experiment were frozen first at -2 C for 24 h. Some of the hatchlings then were allowed to remain at this temperature for another 48 or 72 h whereas animals in other groups were subsequently cooled to minima of -3 (range -2.8 C to -3.2 C) or -4 C (range -3.8 C to -4.2 C). Rates of cooling in the latter tests were 1 C/day or 2 C/day (see Fig. 3). Temperature in the chamber was increased to 2 C at the end of each test and the animals were allowed to thaw overnight. The condition of the turtles (i.e., alive or dead) then was assessed by examining their eyes (which often are only partially open in moribund animals) and by observing their spontaneous activity and respons537 This content downloaded from 207.46.13.129 on Sun, 26 Jun 2016 06:51:49 UTC All use subject to http://about.jstor.org/terms

Temperature during embryonic development irreversibly determines gonadal sex in many oviparous reptiles. Although embryonic temperature also influences a number of other traits in these species, it is unclear whether such effects are primarily due to incubation temperature or to gonadal sex. Here we dissociated these normally confounding effects via hormonal manipulations of snapping turtle embryos (Chelydra serpentina), a species with temperature-dependent sex determination. We then ascertained temperature and sex effects on residual yolk mass, abdominal fat body mass, and total mass in neonates shortly after hatching or after one month without feeding. Yolk mass was initially affected by incubation temperature, clutch identity. and their interaction, but not gonadal sex. Yolk mass was not influenced by any of these variables 30 d after hateching. Fat mass was initially affected by temperature and clutch, but not sex. Gonadal sex did, however, affect fat mass at 30 d of age as did temperature and clutch. Incubation temperature affected total mass in a complex manner. There were significant clutch by temperature interactions initially, but no main effect of temperature. These effects changed so that the temperature effect was significant but interaction effects were not detected at 30 d of age. Repeated measures analysis of total mass after hatching and at 30 d of age indicated that turtles from lower temperatures lost more mass than turtles from higher incubation temperatures. and that females lost more mass than males. These data indicate that some hatchling traits were influenced directly by incubation temperature, whereas others were independently affected by gonadal sex. These results are consistent with a leading hypothesis for the evolution of temperature-dependent sex determination.

Incubation temperature determines sex in the mugger crocodile, Crocodylus palustris . Exclusively females are produced at constant temperatures of 28.0°C through 31°C. At 32.5°C, only males are produced. Both sexes are produced in varying proportions at 31.5, 32.0, and 33.0°C. Embryo survival is not affected within this range, but developmental rate and total incubation time are strongly temperature dependent. In natural nests laid in breeding enclosures, cool incubation temperatures produced only females whereas males were produced only in warm nests. Clutch sex ratios were female or male biased. Yearly sex ratios (=percent male) varied from 0.05 to 0.58; overall sex ratio during six nesting seasons was 0.24 (1 male: 3 females). Sex ratio and incubation time vary with nest location and temperature in a manner consistent with the constant temperature results. Incubation time decreases with increasing incubation temperature, and is an accurate predictor of sex ratio in the field and laboratory. To date, temperature-dependent sex determination (TSD) has been reported in five species of Crocodylus and in three species of Alligatorinae; but the TSD patterns in these groups differ. The TSD pattern of C. palustris is similar to that of C. porosus . Nesting in C. palustris is synchronized with the seasonal availability of thermal regimes suitable for incubation. Resultant sex ratios are a consequence of when and where eggs are laid. Early nests are located in warm, sunny sites; in contrast, late season nests are located in the shade. An egg transplant experiment demonstrated that sex ratios could be altered by simple manipulations of nest temperatures in the field. The adaptive significance of TSD in crocodilians may relate to the influence of incubation temperature on various hatchling attributes, particularly growth.

1. 1. The body temperatures (Tbs) and thermal behavior of hatchling New Guinea crocodiles (Crocodylus novaeguineae) were monitored in a terrestrial-aquatic thermal gradient exposed to the natural photoperiod. 2. 2. At 0–2 weeks of age, hatchlings selected Tbs of 33.4–33.9°C that were significantly elevated by 1.4–2.9°C above levels preferred subsequently. Higher Tbs were pronounced during the period of yolk absorption and may have facilitated the rapid assimilation of unabsorbed yolk immediately after hatching. A similar thermophilic response has been noted for hatchlings of two other species of crocodilians. 3. 3. At 2–5 weeks of age, hatchlings selected lower Tbs (31.8–32.2°C) relative to previous values. Tbs were not elevated or less variable in response to feeding and an increase in heat-seeking behavior was not apparent. 4. 4. Hatchings (2–5-weeks old) preferred Tbs that were approx 2°C higher than the Tbs maintained by a juvenile and adults monitored in a natural setting. Thermal preference in New Guinea crocodiles appeared to decline to adult levels within the first two years of life. 5. 5. Ontogenetic changes in thermal preference, particularly thermophily during early life, are evident in this crocodilian species and may occur in other reptiles as well.

We compare and contrast the signalling systems and social behaviors of Alligator mississippiensis, Crocodylus aculus, and Crocodylus mloticus . Our qualitative analysis focuses primarily on the behaviors of adults during three phases of reproduction: I. Defense of Territory and Courtship, II. Nesting and Incubation, and III. Hatching and Post Hatching. Signals and signal elements are very similar among the three species. For example, all have vocal, non-vocal acoustic, and visual signals, some transmitted through air or water and others through both media. In addition, each species' repertoire is composed of discrete, graded and complex signals. A few signals are unique to each species. However, their signalling systems differ in the temporal organization of the behaviors, and in the relative frequency in which certain functional groups of signals occuror in which signals occur in a particular sensory mode. Apparently, the signalling systems of C. acutus and C. niloticus are more similar to each other than either is to the signalling system of A. mississippiensis . The signalling systems of the crocodile species appear to be adapted to open water habitats in which visual signals are advantageous and to high density breeding groups and post-copulatory intersexual contact. In contrast, the Alligatorsignalling system appears adapted to a marsh habitat in which vocal signals are likely favored and to low density breeding groups.

Juvenile American alligators in outdoor pens moved out of and into the water at sunrise and sunset, respectively. When the natural light cycle was extended with artificial illumination, these movements gradually shifted into phase with the altered light cycles; therefore, the amphibious behavior was modulated by a circadian rhythm cued by light. Movement between land and water was characterized by a decrease in body temperature, which suggests that it is was not simply a proximate heat-seeking response. After the movements had been in phase with the altered light cycles for a time, they spontaneously shifted back into phase with the natural light cycl. A changing response to light is viewed as an adaptation to seasonal changes in heat availability.