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Experimental evidence for the evolutionary significance of temperature-dependent sex determination.

Key words.--Adaptation, antipredator behavior, Chelydra serpentina, directional selection, disruptive selection, selective predation, sex determination, temperature, turtles.

I know your works: you are neither cold nor hot. Would that you were cold or hot! So, because you are lukewarm, and neither cold nor hot, I will spew you out of my mouth. -- Revelation 3:15-16

The origin and significance of sex-determining mechanisms are fundamental problems in evolutionary biology. The sex-determining mechanism of an organism governs the sex ratio of zygotes because it controls the inheritance of sex. In turn, the population sex ratio, a critical demographic parameter, drives the evolution of the sex-determining mechanism (Bull 1983). The expectation that population sex ratio ought to evolve toward unity (Fisher 1930) suggests that those mechanisms producing a 1:1 sex ratio should prevail. However, sex-determining mechanisms vary considerably across the taxonomic spectrum (Bull 1983; Korpelainen 1990) and often yield strongly biased population sex ratios (Bull and Charnov 1988). A major challenge is to explain the evolutionary origin and significance of these atypical sex-determining mechanisms.

Based on their general influence on primary sex ratios, sex-determining mechanisms are divided into two broad categories: genotypic sex determination (GSD) and environmental sex determination (ESD). Most organisms possess some form of GSD in which the sex of the offspring is determined at conception by chromosomal factors. ESD, in which the sex of the offspring is determined by environmental variables after fertilization, occurs in very few, diverse taxa (Bull 1983; Adams et al. 1987; Korpelainen 1990; Janzen and Paukstis 1991a), including rotifers, copepods, and reptiles. Reptiles, in particularly, are an excellent group of organisms with which to investigate the origin and evolutionary significance of sex-determining mechanisms because they exhibit considerable variation in these systems. Although the vast majority of tetrapod vertebrates exhibit some type of GSD, many reptiles have a special case of ESD called temperature-dependent sex determination (TSD) (Bull 1980, 1983; Ewert and Nelson 1991; Janzen and Paukstis 1991a). In the case of TSD, sex of offspring is determined by incubation temperatures experienced during the middle one-third of embryonic development. TSD occurs in all crocodilians, many turtles, and a few lizards (Bull 1980, 1983; Ewert and Nelson 1991; Janzen and Paukstis 1991a,b). Strikingly, various modes of sex determination may be found even among congeneric species. Although evidence is available for other taxa (Bull 1983; Conover 1984; Naylor et al. 1988; Blackmore and Charnov 1989), the enigmas of the ancestry and significance of TSD and GSD in reptiles are currently unresolved. Most investigations of the adaptive significance of TSD in reptiles have searched for fitness components--such as growth rate, body size, or reproductive behavior--that covary with both incubation temperature and offspring sex, but have had little success (Joanen et al. 1987; Gutzke and Crews 1988; Ewert and Nelson 1991; Janzen and Paukstis 1991a,b; Burke 1993). In particular, there have been no satisfactory explanations of this phenomenon in turtles.

To evaluate the adaptive significance of TSD in reptiles successfully using the prevailing theoretical framework, one must establish that (1) incubation temperature influences both offspring gender and individual fitness after hatching, and (2) these posthatching fitness effects are distributed such that a range of temperatures experienced during incubation is better for males than for females and vice versa (Charnov and Bull 1977; Janzen and Paukstis 1991a). In the spirit of this approach, I first empirically addressed the initial requirement of a covariance between individual fitness, gender, and incubation temperature. Subsequently, I obtained preliminary data relevant to the second condition, which requires differential fitness effects of incubation temperature between the sexes. To accomplish these goals, I experimentally integrated the Charnov-Bull model with methods for estimating natural selection (Lance and Arnold 1983; Schluter 1988) to evaluate components of the adaptive significance of TSD in the snapping turtle (Chelydra serpentina) under natural conditions. Body size, locomotor performance, and antipredator behavior of hatchling turtles were evaluated because, as with gender, these traits are influenced by environmental temperatures experienced by developing turtle embryos and, in turn, may be linked to individual fitness (Packard and Packard 1988; Brooks et al. 1991; Janzen 1993a,b).

Materials and Methods

The Study Organism

Snapping turtles (Chelydra serpentina) are very common in North America, occurring in nearly every naturally occurring body of fresh water east of the Rocky Mountains (Ernst and Barbour 1989). These cryptically colored (dark grayish-brown) animals are highly aquatic, typically remaining motionless in shallow water during the day (Hammer 1971; Congdon et al. 1992), although they become more active at night. Snapping turtles are long-lived (>50 yr) (Galbraith and Brooks 1987), highly fecund (11-83 eggs per clutch) (Ernst and Barbour 1989), and do not reach sexual maturity for 11-19 yr (Congdon et al. 1987; Galbraith et al. 1989). During the juvenile period, particularly in the first year of life, most young turtles perish (discussed in Janzen 1993a). A 3-d study of posthatching emergence from the nest and mortality of hatchling snapping turtles in northwestern Illinois found that 41% of the study animals were presumed dead by the end of the observation period (Janzen 1993a). In a South Dakota population of snapping turtles, 3% of hatchlings survived to one year of age and 17% of yearlings survived to 2 yr of age (Hammer 1969). After this time, however, survivorship rates are very high (>93% per year) (Galbraith and Brooks 1987). Consequently, the low survivorship rate of hatchling turtles has an enormous impact on individual lifetime fitness.

Experimental Methods

Collection and Incubation of Eggs.--In June, eggs of common snapping turtles were collected from five freshly constructed nests on National Wildlife Refuge land in Whiteside County, Illinois. Eggs were numbered uniquely with a felttip pen, placed in Styrofoam containers with moistened peat moss, and then transported to the University of Chicago. In the laboratory, eggs were cleaned of adhering soil and were weighed to the nearest 0.01 g. Four eggs from each clutch were randomly assigned to each of eight plastic shoeboxes that contained moistened vermiculite (-150 kPa [nearly equal to] 300 dry vermiculite:337 g deionized water) (Janzen et al. 1990) The 20 eggs in each shoebox were randomly assigned a position within a 4 X 5 matrix. Surplus eggs were placed in four additional shoeboxes as an experimental control group. Substrates in all shoeboxes were rehydrated once weekly to replace water lost by evaporation, thereby providing a constant hydric environment.

To manipulate the sex of the hatchlings, I incubated eggs at an all-male producing temperature (26 [degrees]C), an all-female producing temperature (30 [degrees]C), and an intermediate temperature that produced both sexes (28 [degrees]C). Two shoeboxes each were assigned to incubators calibrated to a constant 26 + 0.5 [degrees]C or 30 [+ or -] 0.5 [degrees]C ; the remaining four shoeboxes were placed in an incubator set at 28 [+ or -] 0.5 [degrees]C. The hydric and thermal conditions used in this experiment are within the range of those measured in natural nests of C. serpentina (Packard et al. 1985). Shoeboxes were rotated daily within the incubators to mitigate the potential influence of thermal gradients on the phenotype, especially sexual differentiation, of developing embryos (Bull et al. 1982).

Hatchling Size and Locomotor Performance.--Upon hatching, turtles were weighed, measured, and then housed under standardized conditions (Janzen 1993a). Two weeks after hatching in each treatment, turtles were evaluated for running and swimming performance at 20 [degrees]C (Janzen 1993a,b). This test temperature was chosen to simulate typical thermal conditions that snapping turtles experience in nature and to facilitate comparison with previous studies (Brown et al. 1990; Janzen 1993a). Sprint speed was measured with a stopwatch for a 1-m interval on a linear racetrack covered with Astroturf. All turtles were stimulated to move by tapping near the tail or rear of the carapace with a forceps. The hatchlings rested for about 75 min between the three trials. Approximately 3 h after tests of running performance were completed, turtles were evaluated for swimming speed over a 1-m interval in a 2-m long x 10-cm wide trough that contained water 5-cm deep. The remainder of the aquatic locomotion procedure was similar to that for the terrestrial locomotion tests. After completion of the races, turtles were marked by clipping a unique combination of marginal scutes on the shell. The mean values for up to three measurements of running and of swimming speed of each individual were used in statistical analyses. Trials in which an individual failed to perform were ignored in calculating these means.

During the performance testing, a number of hatchlings delayed response to stimulation to move and some even refused to run or swim during any of the trials. A quasi-continuous metric was constructed to quantify the propensity to move: 1, performed in zero trials; 2, performed in one trial; 3, performed in two trials; 4, hesitated to perform in all three trials; 5, hesitated to perform in two trials; 6, hesitated to perform in one trial; 7, performed slower than average in all three trials; and 8, performed faster than average in all three trials. This "stubbornness" was interpreted as a potentially informative type of antipredator behavior, because hatchling snapping turtles may prefer to remain cryptically motionless in shallow habitat under natural conditions (Wood 1953; Sexton 1958; Hammer 1971; Congdon et al. 1992). Because many turtles did not perform in all three trials of both running and swimming tests and because the propensity to perform was calculated from the overall behavior for the three trials in each case, the repeatability of these performance and behavior measures could not be reliably assessed. Other ectothermic tetrapod vertebrates, however, including hatchling turtles, exhibit substantial repeatability of performance traits (Bennett 1991; Austin and Shaffer 1992; Janzen 1993b).

Sex Determination.--Sex of the hatchlings was determined by direct examination of gonadal morphology (Janzen 1993b). This procedure was necessary because hatchling C. serpentina, as with offspring of most turtles, do not exhibit sexual dimorphism in external markings, features, coloration, or size. Hatchlings were chilled at -- 20 [degrees]C to achieve immobility prior to the operation. A 5-mm incision was made in the skin anteromedial to the left hind limb and an otoscope was inserted to ascertain the sex. After this procedure, turtles were placed individually into covered containers lined with moistened newspaper. Containers were kept for 3 d at 7 [degrees]C and then for 7 d at 14 [degrees]C. All hatchlings survived the operation. To determine the accuracy of the sexing technique and to test for an effect of the operation on subsequent survivorship, surplus individuals that did not undergo the operation were raised in the laboratory over the winter with a surplus group of otoscopically sexed hatchlings.

Field Methods

To evaluate survivorship under natural conditions, turtles were released into an experimental pond after recovery from the otoscopic examination and were recaptured the following spring. The pond (27-m diameter x 2-m deep) at Kellogg Biological Station (Kalamazoo County, Mich.) is one of 15 similar ponds employed in experimental ecology research projects at the Station (e.g., Werner and Hall 1988). These ponds are similar in dimension to prairie potholes and other naturally occurring bodies of water in which snapping turtles may be found throughout their range (pers. obs.). The pond used in this study was almost completely surrounded by cattails (Typha sp.) and consisted of a thin mud bottom that sloped to a drain (1-[cm.sup.2] mesh) in the center. A drift fence of 20-cm high aluminum flashing was constructed around the pond to prevent potential emigration of hatchlings. Snapping turtles were released into the pond on November 1, and were collected by hand early the following May after the pond was drained. Individuals were identified by the marked marginal scutes. Two subsequent surveys of the pond did not yield any turtles other than those captured in the original census.

Snapping turtles and painted turtles (Chrysemys picta) are often found in the ponds at Kellogg, presumably having migrated from a large swamp adjacent to the pond site (A. Turner, pers. comm. 1991). Red-winged blackbirds (Agelaius phoeniccus), common crows (Corvus brachyrhynchos), gulls (Laws spp.), and a red-tailed hawk (Buteo jamaicensis) were observed in the area when the hatchling turtles were released into the pond. Red-winged blackbirds, common crows, and great blue herons (Ardea herodias) were noticed foraging in the immediate vicinity of or in the pond prior to and during recapture of the turtles in early May. Upon draining the pond, numerous invertebrate larvae and tadpoles were captured, as were bullfrogs (Rana catesbeiana), green frogs (Rana clamitans), red-spotted newts (Notophthalmus viridescens), and two subadult painted turtles. Hence, a variety of predators, prey, and competitors were present during the study, representing an assemblage of species typically encountered by snapping turtles.

Statistical Analyses

Measures of hatchling body size (mass, carapace length, carapace width, plastron length, and head width) and locomotor performance (log of running speed, log of swimming speed, square root of propensity to run, and square root of propensity to swim) were analyzed in SuperANOVA with separate Type III two-way analyses of covariance, using incubation temperature and gender as fixed effects (excluding the interaction term) and initial egg mass as the covariate. Subsequently, a G-test was employed to evaluate the influence of gender and incubation temperature on first-year survivorship (Sokal and Rohlf 1981). Data on survivorship of turtles from the following categories were used to calculate this statistic: 26 [degrees]C, 28 [degrees]C males, 28 [degrees]C females, and 30 [degrees]C. The analyses of covariance and the G-test permit direct tests of the requirement that fitness (or fitness components) must covary with both incubation temperature and gender.

Before statistical investigation of the mechanism of differential survivorship (see Results), the fitness and phenotypic variables used in this analysis were adjusted and transformed in Statview as required by multivariate selection theory (Lance and Arnold 1983). First, relative fitness of each individual was calculated by dividing its absolute fitness by the mean absolute fitness of all the turtles. Juvenile turtles that survived had absolute fitness equal to one, whereas nonsurvivors had absolute fitness equal to zero. Consequently, nonsurvivors were assigned a relative fitness value of zero and survivors were assigned a relative fitness value of 7.57 because approximately 13.2% of the hatchling turtles were recaptured. Five new variables for body size and shape of hatchlings were generated by principal components analysis because the five original measures (see above) were highly intercorrelated. Only the first three principal components (hereafter referred to as PC1, PC2, and PC3) were used in subsequent analyses (see below), because they explained nearly 95% of the original variation in body size and shape of hatchlings. Data for running and swimming performance were adjusted for hatchling size (PC1) using separate linear regressions and then were log-transformed to normalize them. These two performance variables were highly correlated (r = 0.963, P < 0.0001), so only running speed was used in the analysis. Data for running and swimming propensity were Poisson distributed, so they were square root-transformed to improve normality (Sokal and Rohlf 1981). These two measures of propensity to perform were correlated (r = 0.597, P < 0.0001), so only propensity to run was retained for the analysis. Removing these correlated variables reduces the dimensionality of the regression analysis while still permitting determination of the relationship between performance variables and first-year survivorship of the turtles. Measures of running performance were used instead of swimming performance because hatchling snapping turtles, when they move in water, almost invariably crawl (pers. obs.). The means for all variables were set equal to zero before the selection analysis.

The strength and form of natural selection on body size and locomotor performance of hatchling turtles were assessed using multiple quadratic regression analyses in Statview (Lance and Arnold 1983). For the five potential predictors of posthatching survivorship (PC1, PC2, PC3, running performance, and propensity to run), directional selection gradients were first estimated from a multiple regression analysis (Lance and Arnold 1983). Stabilizing selection gradients were then obtained from a subsequent multiple regression analysis that included the five original traits and their squares. Two separate regression analyses are required to estimate the selection gradients, because multivariate nonnormality of the data may otherwise cause the traits to be correlated (Lance and Arnold 1983; Phillips and Arnold 1989). Although estimates of selection gradients from regression analyses are accurate even when fitness components are nonnormally distributed (Lance and Arnold 1983; Brodie and Janzen 1996), significance tests are not immune to this problem. Consequently, the statistical significance of the gradients was evaluated with a delete-one jackknife algorithm (Mitchell-Olds and Shaw 1987). These multiple quadratic regression analyses were used to evaluate morphological or behavioral mechanisms that could explain an observed covariance between individual fitness (i.e., survivorship), incubation temperature, and gender.
TABLE 2. Influence of incubation temperature and gonadal sex on
body size and locomotor performance of hatchling common snapping
turtles (Chelydra serpentina). Values are F-ratios from analyses of
covariance. Error degrees of freedom for log running time and log
swimming time are 106 and 110, respectively.

                                    Source of variation (df)

                                 Temperature   Sex     Egg mass
        Variable                  (2, 116    (1, 116)  (1, 116)

Hatchling mass                     0.89       2.05     489.14(**)
Carapace length                    0.66       0.85     118.02(**)
Carapace width                     0.18       2.98     126.57(**)
Plastron length                    0.21       5.64(*)    0.02
Head width                         0.10       1.54       9.84(**)
Log (running time)                 2.56       0.02      10.50(**)
Log (swimming time)                3.96(*)    2.64       3.86(*)
Square root (running propensity)   5.25(*)    0.06      27.20(**)
Square root (swimming propensity)  1.09       0.13      31.17(**)

(*) P[less than or equal to] 0.05;(**) P [less than or equal to]
0.002.




A cubic spline algorithm (Schluter 1988) was used to visualize the univariate selection surface for significant traits indicated by the regression analyses. Compared to parametric regression, the cubic spline technique is better able to identify dips and modes that might indicate local stabilizing or disruptive selection in an otherwise directional selection surface. Standard errors for the spline analysis were generated from 100 bootstraps of the original data.

Results

Sex Determination,

Overall, 121 of 151 fertile eggs (> 80%) hatched. As expected, all 36 hatchlings from the 26 [degrees]C treatment were males and all 25 hatchlings from the 30 [degrees]C treatment were females. The sex ratio of hatchlings from the 28 [degrees]C treatment was slightly male-biased (37 males and 23 females). The gender of 22 otoscopically sexed surplus hatchlings was evaluated definitively via dissection and matched the diagnoses from the operations in all cases. Furthermore, survivorship of sexed (79%) and unsexed (85%) surplus individuals reared in the laboratory did not differ significantly, indicating that the sexing procedure was not an important source of hatchling mortality (G = 0.059, P > 0.75).

Effects of Temperature and Gender on Morphology, Performance, and Behavior

No measure of body size, locomotor performance, or antipredator behavior of hatchling turtles in the laboratory was significantly affected by both sexual differentiation and incubation temperature (tables 1, 2). One body size variable (plastron length) and two performance traits (log of swimming time and square root of propensity to run) were influenced solely by gender or incubation temperature (tables 1, 2). Upon examination of the raw data, female hatchlings tended to have shorter plastrons than males, but this difference was very small ([nearly equal to] 0.63 mm) and is probably not biologically significant. Also from the raw data, length of time required to swim a 1-m interval rose with increasing incubation temperature: faster turtles derived from cooler incubation treatments (25.4 S at 26 [degrees]C, 35.2 s at 28 [degrees]C, and 42.5 s at 30 [degrees]C). Yet, individuals from 28 [degrees]C had a significantly greater propensity to run than did hatchlings from the other incubation treatments, particularly compared to turtles from 30 [degrees]C (6.22 at 28 [degrees]C vs. 6.17 at 26 [degrees]C and 4.32 at 30 [degrees]C).

Measurement of Natural Selection

Of the 121 hatchling snapping turtles released into the experimental pond, 16 (13.2%) were recaptured the following spring. Consequently, the opportunity for selection, which is equivalent to the variance in relative fitness, was very large (I = 6.617) (Arnold and Wade 1984). Statistical analysis detected significant selection on the interaction of gender and incubation temperature (GINTERACT1ON = 6.622, P = 0.03). Most of the surviving turtles derived from incubation treatments that produced only one sex (26 [degrees]C and 30 [degrees]C) (fig. 1). Turtles from eggs incubated at 28 [degrees]C had significantly lower survivorship than their consexuals from the two extreme temperatures.

[TABULAR DATA 1 OMITTED]

There was no significant directional or stabilizing/disruptive selection on any measure of body size or running performance of hatchling turtles (table 3). Regression analysis of the five traits of interest indicated significant directional selection only on the propensity of hatchling turtles to run (table 3). Individuals that exhibited a tendency to remain motionless in the locomotion tests had significantly higher first-year survivorship than more active turtles (fig. 2). On average, a decrease in propensity to run by one standard deviation would have increased relative fitness by 76% in the population (table 3). The cubic spline analysis did not detect any local stabilizing or disruptive selection within the fitness surface (fig. 2). Hence, first-year survivorship of hatchling snapping turtles in nature covaried with gender and incubation temperature, and the mechanism for this differential survivorship appears to be correlated with the propensity to move.

Discussion

Evolutionary Significance

The evolutionary significance of temperature-dependent sex determination (TSD) in reptiles has defied satisfactory explanation since the discovery of this sex-determining mechanism nearly three decades ago. Despite extensive theoretical work (e.g., Charnov and Bull 1977) and successful demonstrations of the evolutionary significance of environmental sex determination in other taxa (Bull 1983; Conover 1984; Naylor et al. 1988; Blackmore and Charnov 1989), the few empirical studies to address this important question in reptiles have produced inconclusive results and none have previously examined turtles (see discussion below). The work presented herein demonstrates that incubation temperatures clearly influence both gender and posthatching fitness of juvenile snapping turtles. Furthermore, the results of this study indicate that activity behavior of individual turtles may be the mechanism underlying the relationship between incubation temperature, gender, and first-year survivorship of these turtles. Consequently, these preliminary findings are consistent with theoretical conditions required to explain the evolutionary significance of TSD in snapping turtles.

Previous studies of reptiles with TSD have investigated the evolutionary significance of this unusual sex-determining mechanism using two approaches: comparative and experimental. In the former case, Head et al. (1987) first suggested that patterns of sexual dimorphism in adult body size might be related to the mode of sex determination or to the type of TSD in reptiles. This hypothesis was supported by one analysis of available data for reptiles (Ewert and Nelson 1991), but reanalysis of these data using a comparative approach that accounted for phylogenetic relationships was unable to confirm this result (Janzen and Paukstis 1991b). Furthermore, a recent critical review of alternative adaptive explanations rejected group-structured adaptation and sib-avoidance as viable hypotheses for explaining the general occurrence and distribution of TSD in reptiles (Burke 1993).

Experimental studies have enjoyed somewhat greater success in explaining the evolutionary significance of TSD in reptiles. The first such study to address this enigma demonstrated that hatchling alligators (Alligator mississippiensis' exhibited sex-specific posthatching growth rates that were related to their incubation temperatures (Joanen et al. 1987) If these growth rates were to be maintained until adulthood they could translate into differences in reproductive success. that could explain the evolutionary significance of TSD with in the framework of the Charnov-Bull model. Given these results, an ideal experiment would be to raise juvenile alligators to maturity in natural enclosures that permit thermoregulation, because this is an important factor in posthatching growth that is also strongly influenced by incubation temperature (Lang 1987). The only other experimental study to investigate this question in a reptile with TSD examined the effect of incubation temperature on reproductive ability i the leopard gecko (Eublepharis macularius) (Gutzke an Crews 1988). Female geckos from an incubation temperature that produced mostly males did not mate or lay eggs (i.e they were functionally sterile), unlike females from an incubation temperature that produced all females. Unfortunately, this temperature-dependent reproductive ability has yet to be repeated (diets et al. 1993).

Like these experiments, the results of my study provide a solid empirical base from which to launch a more exhaustive experimental investigation of the evolutionary significance of TSD in reptiles. While not definitive, this study indicates that posthatching activity behavior generally predicts firstyear survivorship of hatchling turtles concordant with the interactive effects of gender and incubation temperature (table 3; figs. 1, 2). Despite that result, this experiment can only suggest that some incubation temperatures may be better for one sex than the other, a key requirement of theoretical models on the evolutionary significance of TSD (Charnov and Bull 1977; Bull 1983; Janzen and Paukstis 1991a). Future studies with additional mixed-sex treatments should focus specifically on differentiating between sex-specific and temperature-specific effects on fitness to provide a more definitive explanation for the evolutionary significance of TSD in reptiles. For example, a field experiment on a reptile with TSD showing that the fitness of males from an incubation temperature that produced nearly all males was greater than the fitness of males from an incubation temperature that produced nearly all females, and/or vice versa for females (fig. 3; sensu fig. 2 of Janzen and Paukstis 1991a), would be particularly valuable. Although devising such experiments seems straightforward, in practice identifying satisfactory incubation treatments, fitness components, and the gender of the animals, among other experimental contingencies, have proven difficult to date.

Mechanism of Selection: Predation and Behavior

Selective predation may be the mechanism by which natural selection acted in this experiment. During this study, I observed four separate instances of predation on hatchling snapping turtles: two by bullfrogs (Rana catesbeiana), one by a great blue heron (Ardea herodias), and one by a redwinged blackbird (Agelaius phoeniccus). Gulls (Laws spp.) and common crows (Corvus brachyrhynchos), both known predators on hatchling turtles (Bustard 1979; Walley 1993), were also encountered at the site. All these predatory taxa rely on visual cues to detect prey (Greene 1988).

Body size and running performance, which may be closely linked to predation-related aspects of fitness in hatchling snapping turtles (Janzen 1993a), did not influence survivorship in this study. However, the propensity to run, a likely measure of antipredator behavior, was significantly correlated with first-year survivorship of hatchling snapping turtles (table 3). The more likely a turtle was to exhibit activity in laboratory tests of locomotor performance, the less likely it was to survive in the semi-natural experimental pond (fig. 2). This result is entirely consistent with the natural history of this species (Hammer 1971; Ernst and Barbour 1989; Congdon et al. 1992) and with prior experimental observations (Sexton 1958). Snapping turtles are generally sedentary, I occupying shallow, vegetated habitat; when movement does occur (at least in hatchlings and probably also in adults), the turtles typically crawl along the substrate rather than swim (pers. obs.). Hence, hatchling snapping turtles that remain motionless are cryptic and thus may have increased survivorship over more active individuals (Brooks et al. 1991) caused by decreased predation by visual predators. Experiments with visual predators and hatchling turtles under controlled conditions are needed to test this hypothesis explicitly (e.g., Britson and Gutzke 1993).

Relationships between aspects of performance and survivorship of reptiles under natural conditions have proven somewhat elusive. Two unpublished studies of natural populations of lizards (Sceloporus spp.) (discussed in Bennett and Huey 1990), one study of garter snakes (Thamnophis ordinoides) (Brodie 1992), and one short-term study of hatchling snapping turtles (Janzen 1993a) could not link locomotor performance to survivorship. Only Jayne and Bennett (1990) detected significant selection on locomotor performance in a reptile: garter snakes (T. sirtalis) with a faster burst speed had a greater probability of survivorship. However, Brodie's (1992) experiment and the one reported here have demonstrated significant selection on performance-related aspects of antipredator behavior. Inconsistency in elucidating relationships between performance and fitness in natural populations of reptiles could be attributable to many factors, including phylogenetic effects, ecological irrelevance of certain performance variables, and temporal variation in natural selection. The variation in results among these studies precludes generalizations and therefore invites critical experimental investigation of mechanisms underlying relationships between performance and fitness.

Incubation Temperature and Behavior of Juveniles

Beyond the determination of gender in reptiles with TSD, incubation temperature exerts a physiological influence on many hatchling characteristics (Packard and Packard 1988; Deeming and Ferguson 1991). Statistical analyses indicated that, in addition to gender, propensity of hatchling turtles to run in this study was significantly influenced by incubation temperature (table 2). Hatchlings from 26 [degrees]C and particularly 30 [degrees]C were less likely to run than were turtles from 28 [degrees]C, the treatment with the lowest survivorship (table 1). Indeed, this pattern of behavioral response in relation to incubation history reflects the observations of both gender x treatmentspecific survivorship (fig. 1) and performance-specific survivorship (table 3; fig. 2). Consequently, a likely antipredator behavior of hatchling snapping turtles (i.e., propensity to run) was influenced by incubation temperature and may directly affect survivorship congruent with the results of this study. As previously discussed, additional research is needed to test the functional basis of this hypothesis explicitly.

[TABULAR DATA 1 OMITTED]

Incubation temperature also influences posthatching behavior in several other species of reptiles (Lang 1987; Gutzke and Crews 1988; Burger 1989, 1990, 1991; Van Damme et al. 1992; Janzen 1993b). The findings of Burger's experiments are particularly relevant to the results of this study, because she examined antipredator behavior of hatchling snakes as a function of incubation temperature. Activity scores were lower (Burger 1989) and propensity to burrow or curl up was greater (Burger 1991) for hatchling snakes from low and high incubation-temperature treatments compared to individuals from intermediate incubation temperature environments. In other words, hatchlings that experienced extreme incubation temperatures as embryos were less likely to be active than were snakes that developed in the intermediate temperature treatment. These findings for hatchling snakes mirror the results observed in this study: hatchling turtles from extreme incubation temperatures were less likely to run than were individuals from the intermediate incubation temperature.

The typically dichotomized effects of incubation temperature on gender and behavior in reptiles with TSD may be more strongly linked than is generally recognized. A prominent hypothesis regarding the physiological mechanism of TSD postulates that incubation temperatures directly affect hypothalamic control of gonadotropin releasing hormone (GnRH), which ultimately influences gonadal differentiation through a cascade of hormonal effects (Deeming and Ferguson 1989; reviewed in Janzen and Paukstis 1991a). GnRH and gonadotropin are important in gonadal maturation (Licht 1984; Woods 1987). Electrostimulation of the hypothalamus modifies secretion of gonadal steroids (e.g., Kawakami et al. 1981), which have been directly implicated in gonadal differentiation in reptiles with TSD (reviewed in Crews 1993; Pieau et al. 1994). The hypothalamus also has other well-known physiological functions, influencing thermoregulation, aggression, and motivation (e.g., Berne and Levy 1983). Thus, these behavioral traits, along with gender, may be influenced by incubation temperature through correlated endocrinological pathways in reptiles with TSD (Lang 1987; Gutzke and Crews 1988; this study). Taken together, these facts suggest that behavior, gender, and fitness are strongly linked in reptiles with TSD.

Life-History Implications

The extent to which temperature-dependent antipredator behavior and sex determination of offspring impact lifetime fitness in reptiles with TSD is unknown. First-year survivorship of hatchling snapping turtles in this study was significantly influenced by both factors, but the effects of these characters on reproductive success have yet to be determined. It should be obvious, however, that a turtle cannot reproduce unless it first survives to maturity. A large proportion of hatchling turtles and other reptiles in nature do not emerge from the nest, are eaten shortly after emergence, or do not survive the first winter (Hamilton 1940; Hammer 1969; Burger 1976, 1977; Bustard 1979; Stancyk 1981; Congdon et al. 1987; Charland 1989; Janzen 1993a). Therefore, traits that confer even a modest advantage to hatchling survivorship may experience strong, favorable selection. Hence, natural selection on specific combinations of gender, antipredator behavior, and incubation temperature (figs. 1, 2) may be the evolutionary forces that favored the origin of TSD in turtles and other reptiles, and should greatly affect the subsequent evolution of sex determination in these organisms. Two caveats should be noted however. First, the hatchling snapping turtles in this study were spared the selective sieve of migration from the nests to the pond (e.g., Janzen 1993a), which may favor the evolution of different traits in nature. Furthermore, TSD may have evolved independently several times in reptiles (Janzen and Paukstis 1991a) and different selection pressures could have been involved in each case.

The results reported here also have implications for the selection of nest sites by gravid females, a key aspect influencing the ecology and evolution of TSD (Janzen 1994). The survivorship data from this study imply that the sexual "quality" of offspring is an important component of lifetime fitness. Hatchling turtles derived from incubation treatments that produced only one sex exhibited significantly higher first-year survivorship than hatchlings from the mixed sex treatment (fig. 1). Consequently, male and female hatchlings from nests that produce a mixed sex ratio may have reduced fitness compared to individuals from nests that produce only one sex due to reduced posthatching survivorship. Most nests of species with TSD are unisexual, providing favorable comparative evidence for this hypothesis (reviewed in Ewert and Nelson 1991; Janzen and Paukstis 1991a; Janzen 1994). As with other important issues raised by the present study, more detailed field experiments to test these hypotheses would provide valuable contributions to understanding the origins and evolutionary significance of atypical sex-determining mechanisms.

[Figures 1 to 3 ILLUSTRATION OMITTED]

ACKNOWLEDGMENTS

I thank L. Wargowsky of the U. S. Fish and Wildlife Department for permission to collect turtle eggs, R. and B. Kraciun for temporary housing of eggs, D. Barrick for help in designing the racetrack, and G. Mittelbach and A. Turner at Kellogg Biological Station for their generosity and hospitality. I am grateful to A. M. Bronikowski, D. K. Hews, K. Karoly, H. Landel, and G. L. Paukstis for enlightening discussion of the results, and to J. Altmann, S. J. Arnold, A. F. Bennett, E. D. Brodie III, J. J. Bull, B. Charlesworth, M. Lloyd, M. L. McKnight, L. Rowe, H. B. Shaffer, M. J. Wade, and anonymous reviewers for comments on the manuscript. Turtle eggs were collected under permit W-9225 from the Illinois Department of Conservation. Turtles were treated in accordance with University of Chicago Laboratory Animal Welfare Assurance #52711. This project was supported by a University of Chicago Hinds Fund award, a National Institutes of Health Pre-Doctoral Training Grant in Genetics and Regulation (GM-07197), a National Science Foundation Doctoral Dissertation Improvement Grant (BSR-8914686), and a Postdoctoral Fellowship in the Center for Population Biology at the University of California, Davis. This is Kellogg Biological Station contribution number 780.

(1) Frederic J. Janzen, Present address: Department of Zoology and Genetics, Iowa State University, Ames, Iowa 50011; E-mail: fianzen@iastate.edu.

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Author:Janzen, Fredric J.
Publication:Evolution
Date:Oct 1, 1995
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