Sources and timing of calcium mobilization during embryonic development of the corn snake, Pantherophis guttatus

Embryos of oviparous Reptilia (=turtles, lepidosaurs, crocodilians and birds) extract calcium for growth and development from reserves in the yolk and eggshell. Yolk provides most of the calcium to embryos of lizards and snakes. In contrast, the eggshell supplies most of the calcium for embryonic development of turtles, crocodilians and birds. The yolk sac and chorioallantoic membrane of birds recover and transport calcium from the yolk and eggshell and homologous membranes of squamates (lizards and snakes) probably transport calcium from these two sources as well. We studied calcium mobilization by embryos of the snake Pantherophis guttatus during the interval of greatest embryonic growth and found that the pattern of calcium transfer was similar to other snakes. Calcium recovery from the yolk is relatively low until the penultimate embryonic stage. Calcium removal from the eggshell begins during the same embryonic stage and total eggshell calcium drops in each of the final 2 weeks prior to hatching. The eggshell supplies 28% of the calcium of hatchlings. The timing of calcium transport from the yolk and eggshell is coincident with the timing of growth of the yolk sac and chorioallantoic membrane and expression of the calcium binding protein, calbindin-D28K, in these tissues as reported in previous studies. In the context of earlier work, our findings suggest that the timing and mechanism of calcium transport from the yolk sac of P. guttatus is similar to birds, but that both the timing and mechanism of calcium transport by the chorioallantoic membrane differs. Based on the coincident timing of eggshell calcium loss and embryonic calcium accumulation, we also conclude that recovery of eggshell calcium in P. guttatus is regulated by the embryo.     

Environmentally cued hatching in reptiles

Evidence is accumulating for the widespread occurrence of environmentally cued hatching (ECH) in animals, but its diversity and distribution across taxa are unknown. Herein I review three types of ECH in reptiles: early hatching, delayed hatching, and synchronous hatching. ECH is currently known from 43 species, including turtles, crocodilians, lizards, snakes, tuatara, and possibly worm lizards. Early hatching caused by physical disturbance (e.g., vibrations) is the most commonly reported ECH across all groups; although it apparently serves an antipredator function in some species, its adaptive value is unknown in most. Delayed hatching, characterized by metabolic depression or embryonic aestivation, and sometimes followed by a hypoxic cue (flooding), occurs in some turtles and possibly in monitor lizards and crocodilians; in some of these species delayed hatching serves to defer hatching from the dry season until the more favorable conditions of the wet season. Synchronous hatching, whereby sibling eggs hatch synchronously despite vertical thermal gradients in the nest, occurs in some turtles and crocodilians. Although vibrations and vocalizations in hatching-competent embryos can stimulate synchronous hatching, cues promoting developmentally less advanced embryos to catch up with more advanced embryos have not been confirmed. Synchronous hatching may serve to dilute predation risk by promoting synchronous emergence or reduce the period in which smells associated with hatching can attract predators to unhatched eggs. Within species, advancing our understanding of ECH requires three types of studies: (1) experiments identifying hatching cues and the plastic hatching period, (2) experiments disentangling hypotheses about multiple hatching cues, and (3) investigations into the environmental context in which ECH might evolve in different species (major predators or abiotic influences on the egg, embryo, and hatchling). Among species and groups, surveys for ECH are required to understand its evolutionary history in reptiles. The probability of ECH occurring is likely influenced by a species's life history, ecology, behavior, and interrelationships with other species (e.g., sizes of predator and prey). More broadly, the discovery of embryo-embryo communication as a mechanism for synchronous hatching in crocodilians and turtles indicates that the social behavior of (nonavian) reptiles has been underestimated.     

Trends in the evolution of reptilian chromosomes

Reptiles are a karyologically heterogeneous group, where some orders and suborders exhibit characteristics similar to those of anamniotes and others share similarities with homeotherms. The class also shows different evolutionary trends, for instance in genome and chromosome size and composition. The turtle DNA base composition is similar to that of mammals, whereas that of lizards and snakes is more similar to that of anamniotes. The major karyological differences between turtles and squamates are the size and composition of the genome and the rate at which chromosomes change. Turtles have larger and more variable genome sizes, and a greater amount of middle repetitive DNA that differs even among related species. In lizards and snakes size of the genome are smaller, single-copy DNA is constant within each suborder, and differences in repetitive DNA involve fractions that become increasingly heterogeneous with widening phylogenetic distance. With regard to variation in karyotype morphology, turtles and crocodiles show low variability in chromosome number, morphology, and G-banding pattern. Greater variability is found among squamates, which have a similar degree of karyotypic change-as do some mammals, such as carnivores and bats-and in which there are also differences among congeneric species. An interesting relationship has been highlighted in the entire class Reptilia between rates of change in chromosomes, number of living species, and rate of extinction. However, different situations obtain in turtles and crocodiles on the one hand, and squamates on the other. In the former, the rate of change in chromosomes is lower and the various evolutionary steps do not seem to have entailed marked chromosomal variation, whereas squamates have a higher rate of change in chromosomes clearly related to the number of living species, and chromosomal variation seems to have played an important role in the evolution of several taxa. The different evolutionary trends in chromosomes observed between turtles and crocodiles on the one hand and squamates on the other might depend on their different patterns of G-banding.     

High Evolutionary Rates in Nuclear Genes of Squamates

We compared nonsynonymous substitution rates (Ka) of nuclear coding genes between four major groups of living sauropsids (reptiles): birds, squamates, crocodiles, and turtles. Since only 9 orthologous genes are known in all the four taxonomic groups, we searched for orthologous genes known in chicken and at least one of any representative of poikilotherm sauropsids. Thus, we analyzed three additional data sets: 28 genes identified in chicken and various squamates, 24 genes identified in chicken and crocodilians, and 20 genes identified in chicken and turtles. To compare nonsynonymous substitution rates between all lineages of sauropsids, we used the relative-rate test with human genes as the outgroup. We show that 22/28 nuclear coding genes of squamates, especially snakes (15/16), have an higher evolutionary rate than those in chicken (in mean, 30–40% faster). However, no such difference is detected between crocodiles, turtles and chicken. Higher substitution rate in squamates nuclear coding genes than in chicken, and probably than in other sauropsids, could explain some of the difficulties in resolving the molecular phylogeny of reptiles. Received: 5 July 2000 / Accepted: 13 February 2001     

The family liolopidae (Digenea) including a new genus and two new species from crocodilians

Two new species of liolopid digeneans infecting crocodilians are described and the classification of the family slightly revised. Liolope copulans from the salamander Megalobatrachus japonicus in Japan actually possesses an internal rather than external seminal vesicle and tegumental spines, and the generic diagnosis is emended to include those features. Liolopids paratisitizing crocodilians are removed from Harmotrema and placed in a separate new genus characterized by the possession of a relatively short body with gonads in the posterior rather than middle third of the body. Liolopids infecting Neotropical lizards and turtles remain in Helicotrema and those parasitizing freshwater and marine snakes remain in Harmotrema. The first new species, from Alligator mississipiensis in the southeastern United States, most closely resembles the species described as Harmotrema rudolphii from a saltwater crocodile in the Philippines, but differs from it by having a shorter cirrus sac, stouter and shorter cirrus, and slightly smaller eggs; it differs from all crocodilian liolopids by possessing a more extensively folded metraterm. The second new species, from Crocodylus cataphractis in the Congo (Belgian), differs from all others in crocodilians by having a relatively larger pharynx and smaller acetabulum. Diagnoses of all crocodilian liolopids are presented. Harmotrema laticaudae, previously known from Laticauda laticaudata in Okinawa only, is reported from Aipysurus laevis, Hydrophis major, and a ‘black and white-ringed sea snake’ in Queensland, Australia.     

Sex determination,longevity, and the birth and death of reptilian species

Vertebrate sex‐determining mechanisms (SDMs) are triggered by the genotype (GSD), by temperature (TSD), or occasionally, by both. The causes and consequences of SDM diversity remain enigmatic. Theory predicts SDM effects on species diversification, and life‐span effects on SDM evolutionary turnover. Yet, evidence is conflicting in clades with labile SDMs, such as reptiles. Here, we investigate whether SDM is associated with diversification in turtles and lizards, and whether alterative factors, such as lifespan's effect on transition rates, could explain the relative prevalence of SDMs in turtles and lizards (including and excluding snakes). We assembled a comprehensive dataset of SDM states for squamates and turtles and leveraged large phylogenies for these two groups. We found no evidence that SDMs affect turtle, squamate, or lizard diversification. However, SDM transition rates differ between groups. In lizards TSD‐to‐GSD surpass GSD‐to‐TSD transitions, explaining the predominance of GSD lizards in nature. SDM transitions are fewer in turtles and the rates are similar to each other (TSD‐to‐GSD equals GSD‐to‐TSD), which, coupled with TSD ancestry, could explain TSD's predominance in turtles. These contrasting patterns can be explained by differences in life history. Namely, our data support the notion that in general, shorter lizard lifespan renders TSD detrimental favoring GSD evolution in squamates, whereas turtle longevity permits TSD retention. Thus, based on the macro‐evolutionary evidence we uncovered, we hypothesize that turtles and lizards followed different evolutionary trajectories with respect to SDM, likely mediated by differences in lifespan. Combined, our findings revealed a complex evolutionary interplay between SDMs and life histories that warrants further research that should make use of expanded datasets on unexamined taxa to enable more conclusive analyses.     

Phylogenetic Analysis of Reptilian Hemoglobins: Trees, Rates, and Divergences

Phylogenetic relationships among reptiles were examined using previously published and newly determined hemoglobin sequences. Trees reconstructed from these sequences using maximum-parsimony, neighbor-joining, and maximum-likelihood algorithms were compared with a phylogenetic tree of Amniota, which was assembled on the basis of published morphological data. All analyses differentiated α chains into α^A and α^D types, which are present in all reptiles except crocodiles, where only α^A chains are expressed. The occurrence of the α^D chain in squamates (lizards and snakes only in this study) appears to be a general characteristic of these species. Lizards and snakes also express two types of β chains (βI and βII), while only one type of β chain is present in birds and crocodiles. Reconstructed hemoglobin trees for both α and β sequences did not yield the monophyletic Archosauria (i.e., crocodilians + birds) and Lepidosauria (i.e., Sphenodon+ squamates) groups defined by the morphology tree. This discrepancy, as well as some other poorly resolved nodes, might be due to substantial heterogeneity in evolutionary rates among single hemoglobin lineages. Estimation of branch lengths based on uncorrected amino acid substitutions and on distances corrected for multiple substitutions (PAM distances) revealed that relative rates for squamate α^A and α^D chains and crocodilian β chains are at least twice as high as those of the rest of the chains considered. In contrast to these rate inequalities between reptilian orders, little variation was found within squamates, which allowed determination of absolute evolutionary rates for this subset of hemoglobins. Rate estimates for hemoglobins of lizards and snakes yielded 1.7 (α^A) and 3.3 (β) million years/PAM when calibrated with published divergence time vs. PAM distance correlates for several speciation events within snakes and for the squamate ↔ sphenodontid split. This suggests that hemoglobin chains of squamate reptiles evolved ∼3.5 (α^A) or ∼1.7 times (β) faster than their mammalian equivalents. These data also were used to obtain a first estimate of some intrasquamate divergence times. Received: 15 September 1997 / Accepted: 4 February 1998     

Evolution and Development of Ventricular Septation in the Amniote Heart

During cardiogenesis the epicardium, covering the surface of the myocardial tube, has been ascribed several functions essential for normal heart development of vertebrates from lampreys to mammals. We investigated a novel function of the epicardium in ventricular development in species with partial and complete septation. These species include reptiles, birds and mammals. Adult turtles, lizards and snakes have a complex ventricle with three cava, partially separated by the horizontal and vertical septa. The crocodilians, birds and mammals with origins some 100 million years apart, however, have a left and right ventricle that are completely separated, being a clear example of convergent evolution. In specific embryonic stages these species show similarities in development, prompting us to investigate the mechanisms underlying epicardial involvement. The primitive ventricle of early embryos becomes septated by folding and fusion of the anterior ventricular wall, trapping epicardium in its core. This folding septum develops as the horizontal septum in reptiles and the anterior part of the interventricular septum in the other taxa. The mechanism of folding is confirmed using DiI tattoos of the ventricular surface. Trapping of epicardium-derived cells is studied by transplanting embryonic quail pro-epicardial organ into chicken hosts. The effect of decreased epicardium involvement is studied in knock-out mice, and pro-epicardium ablated chicken, resulting in diminished and even absent septum formation. Proper folding followed by diminished ventricular fusion may explain the deep interventricular cleft observed in elephants. The vertical septum, although indistinct in most reptiles except in crocodilians and pythonidsis apparently homologous to the inlet septum. Eventually the various septal components merge to form the completely septated heart. In our attempt to discover homologies between the various septum components we aim to elucidate the evolution and development of this part of the vertebrate heart as well as understand the etiology of septal defects in human congenital heart malformations.     

Neuroendocrine peptides (insulin, pancreatic polypeptide, neuropeptide Y, galanin, somatostatin, substance P, and neuropeptide gamma) from the desert tortoise, Gopherus agassizii.

The traditional view that Testudines (tortoises and turtles) should be regarded as the surviving clade of the anapsid reptiles rather than classified with the diapsid reptiles (snakes, lizards, and crocodiles) has recently been challenged. Neuropeptide Y, neuropeptide gamma, and somatostatin-14 were isolated from an extract of the brain, substance P and galanin from an extract of the intestine, and insulin and pancreatic polypeptide from an extract of the pancreas of the desert tortoise, Gopherus agassizii. Despite that crocodilians did not appear until the late Triassic, the amino acid sequences of the tortoise peptides resemble those of the American alligator quite closely. The primary structures of neuropeptide Y, somatostatin-14, and neuropeptide gamma are the same in tortoise and alligator. The primary structures of substance P, insulin, galanin, and pancreatic polypeptide in the two species differ by 1, 3, 5, and 8 amino acid residues, respectively. Although fewer neurohormonal peptides from squamates (lizards and snakes) have been characterized, the primary structures of neuropeptide gamma, insulin, and pancreatic polypeptide from the Burmese python and the desert tortoise differ by 3, 8, and 18 residues, respectively. The data suggest, therefore, a closer phylogenetic relationship between Testudines and Crocodilians than that derived from 'classical' analyses based on morphological criteria and the fossil record.     

Explaining large-scale patterns of vertebrate diversity

The major clades of vertebrates differ dramatically in their current species richness, from 2 to more than 32 000 species each, but the causes of this variation remain poorly understood. For example, a previous study noted that vertebrate clades differ in their diversification rates, but did not explain why they differ. Using a time-calibrated phylogeny and phylogenetic comparative methods, I show that most variation in diversification rates among 12 major vertebrate clades has a simple ecological explanation: predominantly terrestrial clades (i.e. birds, mammals, and lizards and snakes) have higher net diversification rates than predominantly aquatic clades (i.e. amphibians, crocodilians, turtles and all fish clades). These differences in diversification rates are then strongly related to patterns of species richness. Habitat may be more important than other potential explanations for richness patterns in vertebrates (such as climate and metabolic rates) and may also help explain patterns of species richness in many other groups of organisms.     

Eggshell Types and Their Evolutionary Correlation with Life-History Strategies in Squamates

The eggshell is an important physiological structure for the embryo. It enables gas exchange, physical protection and is a calcium reserve. Most squamates (lizards, snakes, worm lizards) lay parchment-shelled eggs, whereas only some gekkotan species, a subgroup of lizards, have strongly calcified eggshells. In viviparous (live-bearing) squamates the eggshell is reduced or completely missing (hereafter “shell-less”). Recent studies showed that life-history strategies of gekkotan species differ between species with parchment- and rigid-shelled eggshells. Here we test if the three different eggshell types found in the squamates are also associated with different life-history strategies. We first investigated the influence of the phylogeny on the trait “eggshell type” and on six life-history traits of 32 squamate species. Phylogenetic principal component analysis (pPCA) was then conducted to identify an association between life-history strategies and eggshell types. Finally, we also considered adult weight in the pPCA to examine its potential effect on this association. Eggshell types in squamates show a strong phylogenetic signal at a low taxonomical level. Four out of the six life-history traits showed also a phylogenetic signal (birth size, clutch size, clutches per year and age at female maturity), while two had none (incubation time, maximum longevity). The pPCA suggested an association of life-history strategies and eggshell types, which disappeared when adult weight was included in the analysis. We conclude that the variability seen in eggshell types of squamates is weakly influenced by phylogeny. Eggshell types correlate with different life-history strategies, and mainly reflect differences in adult weights of species.     

Adapting to the unpredictable: reproductive biology of vertebrates in the Australian wet-dry tropics

In the wet-dry tropics of northern Australia, temperatures are high and stable year-round but monsoonal rainfall is highly seasonal and variable both annually and spatially. Many features of reproduction in vertebrates of this region may be adaptations to dealing with this unpredictable variation in precipitation, notably by (i) using direct proximate (rainfall-affected) cues to synchronize the timing and extent of breeding with rainfall events, (ii) placing the eggs or offspring in conditions where they will be buffered from rainfall extremes, and (iii) evolving developmental plasticity, such that the timing and trajectory of embryonic differentiation flexibly respond to local conditions. For example, organisms as diverse as snakes (Liasis fuscus, Acrochordus arafurae), crocodiles (Crocodylus porosus), birds (Anseranas semipalmata) and wallabies (Macropus agilis) show extreme annual variation in reproductive rates, linked to stochastic variation in wet season rainfall. The seasonal timing of initiation and cessation of breeding in snakes (Tropidonophis mairii) and rats (Rattus colletti) also varies among years, depending upon precipitation. An alternative adaptive route is to buffer the effects of rainfall variability on offspring by parental care (including viviparity) or by judicious selection of nest sites in oviparous taxa without parental care. A third type of adaptive response involves flexible embryonic responses (including embryonic diapause, facultative hatching and temperature-dependent sex determination) to incubation conditions, as seen in squamates, crocodilians and turtles. Such flexibility fine-tunes developmental rates and trajectories to conditions--especially, rainfall patterns--that are not predictable at the time of oviposition.     

Complete mitochondrial DNA sequences of the green turtle and blue-tailed mole skink: statistical evidence for archosaurian affinity of turtles.

Turtles have highly specialized morphological characteristics, and their phylogenetic position has been under intensive debate. Previous molecular studies have not established a consistent and statistically well supported conclusion on this issue. In order to address this, complete mitochondrial DNA sequences were determined for the green turtle and the blue-tailed mole skink. These genomes possess an organization of genes which is typical of most other vertebrates, such as placental mammals, a frog, and bony fishes, but distinct from organizations of alligators and snakes. Molecular evolutionary rates of mitochondrial protein sequences appear to vary considerably among major reptilian lineages, with relatively rapid rates for snake and crocodilian lineages but slow rates for turtle and lizard lineages. In spite of this rate heterogeneity, phylogenetic analyses using amino acid sequences of 12 mitochondrial proteins reliably established the Archosauria (birds and crocodilians) and Lepidosauria (lizards and snakes) clades postulated from previous morphological studies. The phylogenetic analyses further suggested that turtles are a sister group of the archosaurs, and this untraditional relationship was provided with strong statistical evidence by both the bootstrap and the Kishino-Hasegawa tests. This is the first statistically significant molecular phylogeny on the placement of turtles relative to the archosaurs and lepidosaurs. It is therefore likely that turtles originated from a Permian-Triassic archosauromorph ancestor with two pairs of temporal fenestrae behind the skull orbit that were subsequently lost. The traditional classification of turtles in the Anapsida may thus need to be reconsidered.     

Reptiles as a food resource

Reptiles have served as an important source of protein for human populations around the world. Exploitation for food is heaviest in the tropical and sub-tropical regions, but also occurs in temperate areas. Of all reptiles, turtles are the most heavily exploited for human consumption. High, unsustainable levels of exploitation for food are directly responsible for the precarious conservation status of many turtles. Crocodilians, snakes, and lizards may be locally important food sources, however, with the exception of a few lizard species, they are exploited in a less intense and generally non-commercial manner for human consumption. In comparison, the commercial skin trade poses a far greater threat to the survival of crocodilians as well as certain large snakes and lizards. Recent field reports have implicated the south east Asian medicinal trade as a growing threat to reptiles, especially turtles and snakes. There are few unequivocal examples of managed harvest programmes for reptiles that are economically and culturally viable, as well as biologically sustainable. Given the economic importance of reptiles as sources of protein and other highly valued commodities, it is imperative that more attention be focused on the development of sustainable use programmes for these species.     

Phylogenetic position of turtles among amniotes: evidence from mitochondrial and nuclear genes

Maximum likelihood analysis, accounting for site-heterogeneity in evolutionary rate with the Gamma-distribution model, was carried out with amino acid sequences of 12 mitochondrial proteins and nucleotide sequences of mitochondrial 12S and 16S rRNAs from three turtles, one squamate, one crocodile, and eight birds. The analysis strongly suggests that turtles are closely related to archosaurs (birds+crocodilians), and it supports both Tree-2: (((birds, crocodilians), turtles), squamates) and Tree-3: ((birds, (crocodilians, turtles)), squamates). A more traditional Tree-1: (((birds, crocodilians), squamates), turtles) and a tree in which turtles are basal to other amniotes were rejected with high statistical significance. Tree-3 has recently been proposed by Hedges and Poling [Science 283 (1999) 998-1001] based mainly on nuclear genes. Therefore, we re-analyzed their data using the maximum likelihood method, and evaluated the total evidence of the analyses of mitochondrial and nuclear data sets. Tree-1 was again rejected strongly. The most likely hypothesis was Tree-3, though Tree-2 remained a plausible candidate.     

Development of cardiac rhythms in altricial avian embryos

Mean heart rate (MHR) was determined during incubation and in hatchlings of 14 altricial avian species to investigate (1) if there is a common developmental pattern of heart rate in altricial embryos and (2) if heart rate changes during incubation are correlated with changes in embryonic growth rate. On the basis of normalized incubation MHR increased approximately linearly in 12 of 14 species from as early as 30-40% of incubation to that of pipped embryos. The MHR of hatchlings was equal to or higher than that of pipped embryos in seven species. Passerine embryos and hatchlings maintained higher MHR in comparison to parrots of similar egg mass, which may reflect phylogenetic differences in development. Embryonic MHR increased at a higher rate while embryonic growth rates were highest during the first 40% of incubation in tit, budgerigar and crow embryos than during subsequent development when relative growth rates decreased. MHR became independent of yolk-free wet mass at a smaller fraction of hatchling mass in budgerigar and crow than in the tit, suggesting that MHR is more likely to increase continuously after 40% of incubation in small altricial species than larger species.     

Rates of projected climate change dramatically exceed past rates of climatic niche evolution among vertebrate species

A key question in predicting responses to anthropogenic climate change is: how quickly can species adapt to different climatic conditions? Here, we take a phylogenetic approach to this question. We use 17 time‐calibrated phylogenies representing the major tetrapod clades (amphibians, birds, crocodilians, mammals, squamates, turtles) and climatic data from distributions of > 500 extant species. We estimate rates of change based on differences in climatic variables between sister species and estimated times of their splitting. We compare these rates to predicted rates of climate change from 2000 to 2100. Our results are striking: matching projected changes for 2100 would require rates of niche evolution that are > 10 000 times faster than rates typically observed among species, for most variables and clades. Despite many caveats, our results suggest that adaptation to projected changes in the next 100 years would require rates that are largely unprecedented based on observed rates among vertebrate species.     

Morphometric analysis of embryonic development in Alligator mississippiensis, Crocodylus johnstoni and Crocodylus porosus

The size of embryos at various stages of development was determined in three species of crocodilian ( Alligator mississippiensis, Crocodylus johnstoni and C. porosus). Various morphometric measurements were taken of embryos throughout development and were described for each stage of development. Increase in size from stage to stage was faster in A. mississippiensis than in C. porosus and C. johnstoni. Hatchlings of A. mississippiensis were large in length but light in mass compared with the hatchlings of C. johnstoni and C. porosus which were heavier per unit length. These morphometric parameters can be used to determine the stage of embryonic development by size. The use of principle component analysis improves this technique further by dampening any anomalous data points.
The rate of embryonic growth in A. mississippiensis appeared to be under greater genetic control than in the two species of Crocodylus. The evolutionary advantages of this phenomenon probably relate to the biology of A. mississippiensis. Due to the northerly range of this species it is advantageous for alligators to hatch as soon as possible, as large as possible, to maximize the period prior to winter hibernation and reduce predation. Tropical crocodiles have fewer selection pressures for rapid development and have slower rates of embryonic growth. Genetic aspects of crocodilian embryonic development have been largely ignored but may help explain some aspects of crocodilian growth under farming conditions.     

Environmental regulation of sex determination in reptiles

The various patterns of environmental sex determination in squamates, chelonians and crocodilians are described. High temperatures produce males in lizards and crocodiles but females in chelonians. Original experiments on the effects of incubation at 30 degrees C (100% females) or 33 degrees C (100% males) on development in Alligator mississippiensis are described. These include an investigation of the effect of exposing embryos briefly to a different incubation temperature on the sex ratio at hatching, and a study of the effects of 30 degrees C and 33 degrees C on growth and development of alligator embryos and gonads. A 7-day pulse of one temperature on the background of another was insufficient to alter the sex ratio dramatically. Incubation at 33 degrees C increased the rate of growth and development of alligator embryos. In particular, differentiation of the gonad at 33 degrees C was enhanced compared with 30 degrees C. A hypothesis is developed to explain the mechanism of temperature-dependent sex determination (TSD) in crocodilians. The processes of primary sex differentiation are considered to involve exposure to a dose of some male-determining factor during a specific quantum of developmental time during early incubation. The gene that encodes for the male-determining factor is considered to have an optimum temperature (33 degrees C). Any change in the temperature affects the expression of this gene and affects the dose or quantum embryos are exposed to. In these cases there is production of females by default. The phylogenetic implications of TSD for crocodilians, and reptiles in particular, are related to the life history of the animal from conception to sexual maturity. Those animals that develop under optimal conditions grow fastest and largest and become male. A general association between the size of an animal and its sex is proposed for several types of vertebrate.