Michael A. Ewert

Sex Determination in Turtles: Diverse Patterns and Some Possible Adaptive Values

New data on the genders of young turtles from eggs incubated at controlled temperatures demonstrate temperature-dependent sex determination (TSD) in 17 species surveyed for the first time and corroborate TSD in another 11 species. The well-known pattern of males from cool temperatures and females from warm ones (=Pattern Ia) occurs in eight species and may occur in seven more. A pattern of cool females, intermediate males, and warm females (=Pattern II) occurs in ten species. Data on three species surveyed for the first time are compatible with genetic sex determination (GSD). Within Clemmys, C. guttata has TSD; C. insculpta, GSD. Comparisons among phylogenetic sister groups suggest at least four independent losses of TSD in turtles. Large variation in extent of sexual differentiation at hatching among TSD species vs GSD species obscures any evidence that either system provides earlier or more complete differentiation. Pattern Ia occurs mainly in species in which adult females average larger than adult males; Pattern II occurs mainly in species with females smaller than males or in which body size is not dimorphic. Among TSD reptiles generally, the smaller gender typically arises at the coolest incubation temperatures. The new data, together with a literature survey, invite four possible explanations for various aspects of sex determination in reptiles: phylogenetic inertia, temperature-dependent differential fitness, sib-avoidance, and group-structured adaptation in sex ratios. Key demographic features from the literature include a high incidence of unisexuality within clutches and a predominance of female biases in sex ratios of hatchlings but not in those of adults. Each of these explanations remains partly but not fully plausible. Explanations for the patterns of sex determination may ultimately require a combination of hypotheses.

Environmental sex determination in a reptile varies seasonally and with yolk hormones

Most hypotheses that have been put forward in order to explain the persistence of environmental sex determination (ESD) in reptiles assume a relatively fixed association of sex with temperature–induced phenotype and no maternal influence on offspring sex. Here we demonstrate the association of maternally derived yolk hormone levels with the offspring sex ratio and describe two new aspects of temperaturedependent sex determination (TSD), i.e. seasonal variation in both thermal response and yolk steroid levels. Eggs from painted turtles (Chrysemys picta) were incubated at 28°C. The hatchling sex ratio at 28°C (i.e. the phenotypic reaction norm for sex at 28°C) shifted seasonally from ca. 72% male to ca. 76% female. Yolk oestradiol (E2) increased seasonally while testosterone (T) decreased. The proportion of males in a clutch decreased as E2 levels increased and the E2:T ratio increased. These new findings are discussed in relation to heritability and adaptive explanations for the persistence of ESD in reptiles. Maternally derived yolk hormones may provide a mechanism for the seasonal shift in the sex ratio which in turn may help explain the persistence of ESD in reptiles. They may also explain those clutches of other reptiles with TSD that fail to yield only males at maximally masculinizing conditions.

Nesting fidelity and molecular evidence for natal homing in the freshwater turtle, Graptemys kohnii

Numerous studies of sea turtle nesting ecology have revealed that females exhibit natal homing, whereby they imprint on the nesting area from which they hatch and subsequently return there to nest as adults. Because freshwater turtles comprise the majority of reptiles known to display environmental sex determination (ESD), the study of natal homing in this group may shed light on recent evolutionary models of sex allocation that are predicated on natal homing in reptiles with ESD. We examined natal homing in Graptemys kohnii, a freshwater turtle with ESD, using mitochondrial sequencing, microsatellite genotyping and mark and recapture of 290 nesting females. Females showed high fidelity to nesting areas, even after being transplanted several kilometres away. A Mantel test revealed significant genetic isolation by distance with respect to nesting locations (r=0.147; p<0.05), suggesting that related females nest in close proximity to one another. The patterns of fidelity and genotype distributions are consistent with homing at a scale that may affect population sex ratios.

Hormone levels in yolk decline throughout development in the red-eared slider turtle (Trachemys scripta elegans)

This study investigates the potential effects of maternally derived hormones present in the yolk of reptile eggs. Specifically, we ask when are these hormones utilized by developing red-eared slider turtles (Trachemys scripta elegans), a species with temperature-dependent sex determination (TSD). Eggs were incubated at 27 degrees C, a male-producing temperature, and at 31 degrees C, a female-producing temperature. Concentrations of progesterone, testosterone, and 17beta-estradiol were measured at four points during development: at oviposition, at the start of the temperature sensitive period (TSP), at the end of the TSP, and at hatching. No effects of incubation temperature on yolk hormone concentrations were detected. The highest concentrations of all three hormones were measured at oviposition. Hormone-specific patterns of decline occurred throughout development. Each hormone declined between oviposition and the early TSP. Although estradiol was present in detectable quantities at oviposition, it was virtually undetectable by the early TSP. Testosterone showed no further decline after the early TSP. Progesterone continued to decline between the early and post-TSP. These results demonstrate that maternally derived yolk hormones decline at different rates. Alternative explanations for the disappearance of these yolk hormones are presented.

Long-term sex reversal by oestradiol in amniotes with heteromorphic sex chromosomes

Oestradiol application during embryonic development reverses the sex of male embryos and results in normal female differentiation in reptiles lacking heteromorphic sex chromosomes, but fails to do so in birds and mammals with heteromorphic sex chromosomes. It is not clear whether the evolution of heteromorphic sex chromosomes in amniotes is accompanied by insensitivity to oestradiol, or if the association between oestradiol insensitivity and heteromorphic sex chromosomes can be attributable to phylogenetic constraints in these taxa. Turtles provide an ideal system to examine the potential relationship between oestradiol insensitivity and sex chromosome heteromorphy, since there are species with heteromorphic sex chromosomes that are closely related to species lacking heteromorphic sex chromosomes. We investigated this relationship by examining the long-term effects of oestradiol-17β application on sex determination in Staurotypus triporcatus and Staurotypus salvinii, two turtle species with male heterogamety. After raising the turtles in the lab for 3 years, we found follicular and Müllerian duct morphology in oestradiol-treated turtles that was identical to that of untreated females. The lasting sex reversal suggests that the evolutionary transition between systems lacking heteromorphic sex chromosomes and those with heteromorphic sex chromosomes is not constrained by a fundamental mechanistic difference.

Incubation Environment Affects Immune System Development in a Turtle with Environmental Sex Determination

Abstract The developmental environment can have lasting effects on posthatching phenotype in oviparous animals. Innate immune response is one important component of fitness in vertebrates because it provides a generalized defense against infection. In addition, because male vertebrates are at a higher risk of infection than females, males may benefit more from increased innate immunity than females. We determined the effects of incubation temperature on the innate immune response of hatchling map turtles (Graptemys) by incubating eggs at a range of male and female producing-temperatures and assessing plasma complement activity in the resulting hatchlings. We found a significant effect of incubation environment on circulating complement in hatchling Graptemys ouachitensis, with male-producing temperatures yielding the highest innate immune response. Most important, these results demonstrate that immune response is affected by developmental environment in a species with environmental sex determination, potentially resulting in sex differences in the ability to fend off pathogens.

Sex Determination and Ontogeny in Malacochersus tornieri, the Pancake Tortoise

Abstract We document temperature-dependent sex determination (TSD) in the Pancake Tortoise (Malacochersus tornieri). The nesting season is biased toward a fall–winter pattern and the embryos have a postovipositional developmental arrest. Males initially grow faster than females, but females are the larger sex. The species does not readily conform to a hypothesis that TSD is adaptively maintained through a direct association of juvenile growth with adult sexual size dimorphism.

Estradiol-17β Induces Lasting Sex Reversal at Male-Producing Temperatures in Kinosternid Turtles

Abstract In turtles with temperature-dependent sex determination (TSD), estradiol applied to eggs incubated at male-producing temperatures produces female hatchlings. In other vertebrate taxa, estradiol administration during embryogenesis often induces only temporary or incomplete feminization, and sensitivity to estradiol can vary among taxonomic families. In the present study, we treated eggs of Kinosternon scorpiodes and Sternotherus odoratus incubated at male-producing temperatures with estradiol-17β and examined reproductive morphology of offspring. Furthermore, to determine whether sex reversal resulting from estradiol application in turtles with TSD is lasting, we maintained juvenile K. scorpiodes in the lab for up to three years prior to dissection. We found normal ovarian morphology and Müllerian duct development in a significantly greater proportion of the hatchling S. odoratus and juvenile K. scorpiodes that had been treated with estradiol during embryogenesis than in the untreated group. Results further suggest that estradiol-induced sex reversal seen in TSD turtles is both permanent and ubiquitous and may shed light on the evolution of sex-determining mechanisms in vertebrates.

Reproductive Trends in Captive Heosemys grandis (Geoemydidae)

Abstract A 20-year record of captive breeding of a female Heosemys grandis revealed a tradeoff between egg size and clutch size across the years when she produced 2 clutches per breeding season. First clutches had few large eggs and second clutches had a large number of smaller eggs. Four F1 progeny from this founder female began their reproductive years with much smaller eggs; however, their eggs increased in size over successive years until they were the same size as those of the long-term breeder.

Carbon dioxide influences environmental sex determination in two species of turtles

Eggs of wild caught Trachemys scripta and Graptemys pseudogeographica kohnii , two emydine turtles known to have temperature-dependent sex determination, were incubated at 28.5°C in boxes aerated with one of four gas mixtures (two in G. p. kohnii ). Across all eggs, elevated levels of carbon dioxide significantly feminized sex ratios in both species and also reduced hatching success. When eggs were grouped into clutches, all comparisons between 0% and elevated carbon dioxide showed feminization in the portions of the clutches at elevated carbon dioxide, a statistically significant result for T. scripta. Accumulation of biogenic carbon dioxide from embryonic respiration and other sources is thus likely to affect sex ratios in natural nests under some conditions, perhaps through an effect on some aspects of embryonic pH.