Phenotypic variation in smooth softshell turtles (Apalone mutica) from eggs incubated in constant versus fluctuating temperatures

Oecologia (2003) 134:182 188 DOI 10.1007/s00442-002-1109-z ECOPHYSIOLOGY Grant M. Ashmore Fredric J. Janzen Phenotypic variation in smooth softshell turtles (Apalone mutica) from eggs incubated in constant versus fluctuating temperatures Received: 15 January 2002 / Accepted: 15 October 2002 / Published online: 26 November 2002 Springer-Verlag 2002 Abstract Temperatures experienced during embryonic development elicit well-documented phenotypic variation in embryonic and neonatal animals. Most research, however, has only considered the effects of constant temperatures, even though developmental temperatures in natural settings fluctuate considerably on a daily and seasonal basis. A laboratory study of 15 clutches of smooth softshell turtles (Apalone mutica) was conducted to explicitly examine the influence of thermal variance on phenotypic variation. Holding mean temperature constant and eliminating substrate moisture effects permitted a clear assessment of the impact of thermal variance on hatching success, incubation length, hatchling body size, swimming speed, and righting time. Incubation length and swimming speed varied significantly among temperature treatments. Both traits tended to increase with increasing thermal variance during embryonic development. Clutch significantly affected all traits examined, except righting time, even after accounting for the effects of initial egg mass. These results highlight the importance of accounting for the impact of both thermal mean and variance on phenotypic variation. The findings also strengthen the increasing recognition of maternal clutch effects as critical factors influencing phenotypic variation in neonatal animals. Keywords Egg Embryo Hatchling Temperature Turtle Introduction Abiotic factors exert fundamental impacts on developing embryos. In a wide variety of taxa, developmental temperature in particular can affect numerous key G.M. Ashmore F.J. Janzen () ) Department of Zoology & Genetics, Iowa State University, Ames, IA 50011-3223, USA e-mail: fjanzen@iastate.edu Tel.: +1-515-2944230 Fax: +1-515-2948457 ecological traits of offspring, including development rate, pigmentation pattern, body size, growth, behavior, and even sexual differentiation (e.g., Ratte 1985; Deeming and Ferguson 1991; Burggren and Just 1992; Schrag et al. 1994; Billerbeck et al. 2000). Although such well-known ecological phenomena are often underappreciated in the current genomic age, their frequent rediscovery by geneticists illustrates the perils involved in ignoring the important role of the environment in development (e.g., Pennisi 2002). Environmental effects on developing reptile embryos have been well established in the last few decades. Survivorship and phenotypic attributes are influenced by differing environmental conditions (reviewed in Deeming and Ferguson 1991). Among those species that lay flexible-shelled eggs, both temperature and substrate moisture typically play a large part in affecting hatchling traits (reviewed in Packard and Packard 1988). For example, incubation on relatively wet substrates tends to cause turtle eggs, embryos and, subsequently, hatchlings to be larger in size than those eggs kept on drier substrates. Moisture also influences the length of incubation and hatching success (Packard and Packard 1988), as well as locomotor performance of neonates (Miller et al. 1987). In contrast, species that lay rigid-shelled eggs tend to be affected by incubation temperature (Janzen 1993b) but less so by water content in the soil (Leshem and Dmi el 1986). Substrate moisture seems not to affect embryonic metabolism or hatchling mass (Packard et al. 1979, 1981; Packard and Packard 1991). Consequently, these species are excellent models for examining the influence of temperature on phenotypic variation of hatchlings without the potentially confounding effects of substrate moisture. Different incubation temperatures elicit well-documented variation in a number of traits in embryonic and hatchling reptiles (e.g., Ewert 1979; Deeming and Ferguson 1991; Janzen 1993b). At the same time, these findings may not reflect what truly occurs in a natural setting. Temperatures are kept constant throughout incubation in most studies, yet it is well known that temperature within

a reptile nest fluctuates on a daily basis (e.g., Plummer et al. 1994; Shine and Harlow 1996; Valenzuela 2001). How and whether fluctuating temperatures influence developing reptile embryos differently than constant temperatures with the same mean is largely unknown (Georges et al. 1994; Overall 1994; Shine and Harlow 1996; Shine et al. 1997; Doody 1999; Andrews et al. 2000; Webb et al. 2001), except for extensive work on temperature-dependent sex determination in turtles (most recently discussed in Valenzuela 2001). In this study, we evaluate the influence of constant versus fluctuating incubation temperatures (holding the mean constant) on hatching success, body size, and performance of hatchlings of the smooth softshell turtle, Apalone mutica (LeSueur 1827). Unlike many turtles, softshells (Trionychidae) lay rigid-shelled eggs (Fitch and Plummer 1975; Ernst et al. 1994); thus, substrate moisture can be eliminated as a major causative agent leading to developmental perturbation. Consequently, phenotypic variation detected in different temperature regimens in A. mutica can be confidently assigned to such thermal treatments after clutch effects have been taken into account. Materials and methods Egg collection and treatment Eggs of A. mutica were collected from 15 natural nests on a sandbar in the Cedar River near Muscatine, Iowa, USA on 10 June 2000. All eggs were presumed to have been laid within 2 days of collection as indicated by either the presence of a small chalk spot (i.e., beginning of an air space) or no spot at all (Ewert 1979, 1985; Plummer et al. 1994). Eggs were individually marked in the field and the majority were transported to the laboratory in Ames, Iowa. Forty-five eggs, three chosen randomly from each clutch, were weighed in the field and reburied to study natural phenotypic variation. Data beyond initial mass and count of these eggs are not included in this report as the sandbar flooded twice during incubation and drowned all but two of the embryos in the companion field experiment. In the laboratory, eggs were brushed free of sand and were weighed to the nearest 0.01 g. At least one egg from each of 13 clutches was arranged randomly in a 3 6 matrix in each of 9 covered plastic boxes containing moistened vermiculite (300 g dry vermiculite: 337 g water» 150 kpa) (Janzen et al. 1990). For the remaining two clutches, which had counts of 10 and 11 eggs, one egg was arranged randomly in each of 7 and 8 of the 9 covered plastic boxes, respectively. Water was added to the boxes once each week throughout incubation according to weight lost by evaporation. Three boxes were placed in each of three incubators: one kept at a constant 30.5C, another kept at 30.5 2C, and a third kept at 30.5 4C. These conditions were confirmed with HOBO-TEMP temperature loggers (Onset Computer, Pocasset, Mass.) (Fig. 1). Fluctuation was maintained at 12 h (+) and 12 h ( ). Boxes were rotated within each incubator every other day to minimize potential thermal gradients. Unfortunately, treatments could not be replicated for logistic reasons and are thus confounded with incubators. Nonetheless, the fidelity of the temperature records, the clear differences between the treatments (Fig. 1), and the similarity in results between the constant 30.5C treatment and prior research using a similar constant temperature (Janzen 1993b) lend confidence in interpreting the results of this experiment in the context of thermal, rather than incubator, effects (see also Packard and Packard 1993). The mean temperature of 30.5C was chosen based on higher survival rates found in prior studies of A. mutica at 30C (Ewert 1979; Janzen 1993b; Plummer et al. 1994). Fluctuation was chosen to be maximal at 4C because embryonic development below 26.5C might be inhibited (Janzen 1993b). Temperature fluctuations at 12-min intervals measured in the center of nests (~20 cm below the surface) at the collection site in summer 2000 (via HOBO-TEMP temperature loggers) confirmed the laboratory treatments to be somewhat stepped but reasonably similar to natural nest conditions (Fig. 1). Treatment effects were not considered here in the context of constant temperature equivalents (sensu Georges et al. 1994) because the approach has not (yet) been widely validated (e.g., Doody 1999). Hatching success and days to pip Immediately after an egg was discovered to have pipped, a bottomless plastic cup was placed around the egg to confine the turtle for correct identification. Dates of pipping and hatching were recorded and rarely differed by more than a few hours for each hatchling. Days to pip (i.e., duration of incubation) was analyzed by two-way ANOVA with incubation treatment as a fixed factor, and clutch and the interaction between clutch and incubation treatment as random effects. Initial egg mass was tested as a potential covariate but was found to have no significant effect (P=0.22) and was thus discarded. All ANOVAs and ANCOVAs were performed using Proc Mixed in SAS v. 6.12. Significance levels for variance components for random effects were estimated using the test option in Proc GLM. Only P-values are thus reported for all ANOVAs and ANCOVAs in this paper for simplicity. Body size 183 Fig. 1 Temperature profiles for a 2-week period for one Apalone mutica nest on a sandbar in the Cedar River and three incubationtemperature treatments used in the laboratory during the summer of 2000. Daily temperature fluctuations in nests were substantial (occasionally >10C) except when nests were flooded (e.g., 24 25 June). Overall, egg-incubation conditions in the laboratory were a reasonable reflection of the mean and variance of nest temperatures Two days after hatching, turtles were washed free of vermiculite, towel dried, and then weighed to the nearest 0.01 g. Immediately thereafter, midline carapace length, carapace width at midbody, and plastron length were measured to the nearest 0.1 mm with dial calipers. Intercorrelations among measures of body size were calculated using Statview v. 1.02. Analyses of covariance, with initial egg mass as the covariate, were performed to evaluate the effects of

184 incubation treatment (fixed), clutch (random), and the interaction between clutch and incubation treatment (random) on the various measures of body size. ANCOVA provides some control for maternal effects associated with egg mass that could otherwise be confounded with different clutch effects (Garland 1984, 1988; Garland and Bennett 1990; Janzen 1993a). Performance Hatchlings were housed individually in covered, though not sealed, plastic cups with overly saturated paper towels at 23C. Though previous studies of A. mutica involved keeping hatchlings in 2 cm tap water prior to performance testing (Janzen 1993b), this arrangement was avoided until after testing in this study. That is, the fitness value of performance may be best revealed at the time of initial exposure to an environment, such as a terrestrially hatched neonate first encountering its permanent aquatic home (e.g., Wyneken and Salmon 1992). Swimming speed was evaluated immediately after body-size measurements were recorded. Three swim tests were performed for each hatchling with a 30-min rest between trials. Swimming speed for a 1-m interval was measured in a 1.2-m 10-cm trough containing water 2.5 cm deep. Hatchlings were stimulated to move by tapping the trough just behind their tails with forceps to simulate predatory attacks by birds. Although a few hatchlings refused to swim with this prompting, no physical contact between the forceps and hatchling occurred. Swimming speed was calculated from the mean of all trials completed by an individual. Thirty minutes after completion of the swim trials, flip tests were performed. Each hatchling was turned over on its back on a dry paper towel on a level surface and given a maximum of 60 s to right itself. A 15-s resting period was permitted between each flip. Righting time was determined from the average of five flip trials. Two-way ANOVAs were used to evaluate the influence of incubation treatment as a fixed factor, and clutch and the interaction between clutch and incubation treatment as random effects on mean swimming speed and mean righting time. Hatchling mass and carapace length were tested as potential covariates but were found to have no significant effect on either swimming speed or righting time (P>0.21 in all cases) and were thus discarded. All turtles were released in the Cedar River at the site of egg collection on 1 September 2000. Results Eggs The mass of eggs ranged from 5.36 to 7.69 g with an average of 6.29 0.50 g ðx SDÞ. Clutch size was as small as 10 and as large as 20 and averaged 13.5 2.4 eggs ðx SDÞ. Total clutch masses ranged from 66.25 to 130.98 g with an average of 84.72 16.00 g ðx SDÞ. These results are consistent with those observed for this same population of A. mutica in 1989 and 1990 (Janzen 1993b): egg masses of 6.41 0.60 g (1989) and 6.55 1.07 g (1990), clutch sizes of 14.0 4.5 (1989) and 13.1 3.2 (1990), and clutch masses of 89.68 30.77 g (1989) and 86.83 22.94 g (1990). Hatching success and days to pip Hatching success of fertile eggs, which was extremely high, was not affected by incubation treatment (Table 1). Incubation temperature treatment and clutch, though not Table 1 Statistics for hatching success, days to pip, egg and hatchling measurements, and performance as a function of incubation-temperature treatment. Values (other than hatching success and initial egg mass) are least-squares means SEs. Data for the four body-size variables were adjusted by ANCOVA to remove the effect of initial egg mass. Sample sizes are given in parentheses above each column with the following exceptions: n=44 for days to pip from 30.5 2C; n=44 and n=45 for swimming speed from 30.5 2C and 30.5 4C, respectively; n=39, n=42, and n=43 for righting time from 30.5C, 30.5 2C, and 30.5 4C, respectively Variable 30.5C 30.5 2C 30.5 4C (n=44) (n=45) (n=46) Hatching success (%) 100 95.7 97.9 Days to pip (days) 52.3 0.2 51.3 0.2 54.4 0.2 Initial egg mass (g) 6.3 0.5 6.3 0.5 6.3 0.4 Hatchling mass (g) 4.3 0.03 4.3 0.03 4.4 0.03 Carapace length (mm) 35.7 0.2 35.5 0.1 35.8 0.2 Carapace width (mm) 31.4 0.1 31.4 0.1 31.5 0.1 Plastron length (mm) 25.6 0.1 25.6 0.1 25.3 0.1 Swimming speed 7.8 0.7 8.9 0.7 11.7 0.7 (cm* s 1 ) Righting time (s) 0.9 0.3 1.1 0.3 1.2 0.3 the interaction between the two, influenced the number of days to pip (P<0.0001 for both temperature and clutch and P=0.90 for their interaction). Developmental duration was shortest in the 30.5 2C treatment and longest in the 30.5 4C treatment, with the constant 30.5C in between (Table 1). Post-hoc tests indicated that all treatments differed significantly from each other (P 0.0003 in all three pairwise comparisons). Body size All measures of hatchling body size were significantly and positively correlated with initial egg mass. Additionally, these measures were highly intercorrelated (Table 2), indicating that larger hatchlings tended to hatch from larger eggs. Consequently, initial egg mass was used as a covariate in statistical analyses of the effect of incubation temperature and clutch on body size. Little variation was detected within each measure of body size across the three temperature conditions (Table 1): hatchlings were similar in size regardless of incubation treatment (P 0.17 in all four cases). There were also no significant treatment by clutch interactions (P 0.17 in all four cases). In contrast, clutch explained significant variation in all four measures of hatchling size (P 0.05 in all four cases). Performance Swimming speed was positively correlated with all measures of body size. In contrast, righting time was not significantly correlated with any measure of body size (Table 2). Not surprisingly, there was also no significant correlation between the two measures of performance. Swimming speed was strongly affected by temperature treatment and clutch (P 0.0005 in both cases), but not by

Table 2 Correlations among size and performance measurements; n=135 with the following exceptions: n=134 for correlations involving days to pip; n=133 for correlations involving swimming Variable Initial egg mass Hatchling mass Carapace length speed; and n=124 for correlations involving righting time. P 0.01 except as indicated Carapace width Plastron length Swimming speed Righting time Days to pip.130 b.215.272.238.053 b.421.190 a Initial egg mass 1.746.685.643.633.226.041 b Hatchling mass 1.867.822.774.282.013 b Carapace length 1.815.725.160 a.040 b Carapace width 1.708.178 a.105 b Plastron length 1.283.001 b Swimming speed 1.074 b a 0.02 P 0.07 P 0.14 185 their interaction (P=0.96). The greater the temperature fluctuation, the greater the swimming speed (Table 1). More specifically, post-hoc tests indicated that hatchlings from the constant 30.5C and 30.5 2C treatments were significantly different from (i.e., slower than) hatchlings from the 30.5 4C treatment (P<0.01 in both cases). Swimming speeds of turtles from the constant 30.5C and 30.5 2C treatments were not significantly different from each other, however (P=0.27). Righting time was not significantly influenced by temperature treatment, clutch, or their interaction (P 0.09 in all three cases). Even so, righting time tended to increase with increasing temperature fluctuation (Table 1). Discussion Incubation temperature has dramatic effects on eggs, embryos, and offspring of oviparous organisms (Ewert 1979; Ratte 1985; Deeming and Ferguson 1991). Nonetheless, these thermal impacts have largely been characterized on the basis of constant temperatures, although temperatures during embryogenesis fluctuate considerably in nature. Research on temperature-dependent sex determination in reptiles has increasingly recognized the importance of this thermal variation (e.g. Valenzuela 2001). However, with the exception of a rich literature on insects (reviewed in Ratte 1985; Hagstrum and Milliken 1991), only a handful of experiments (on lizards, snakes, and turtles) have explicitly evaluated the impact of thermal variance per se on eggs, embryos, and offspring (Georges et al. 1994; Overall 1994; Shine and Harlow 1996; Shine et al. 1997; Doody 1999; Andrews et al. 2000; Webb et al. 2001). In general, they find that fluctuating incubation temperatures elicit significant variance in many phenotypic traits that often differs from variance obtained using constant temperatures even under identical mean temperatures. In our study, temperature fluctuation had a profound effect on incubation length and swimming speed in a turtle. Our results are particularly insightful because, with the exception of Doody (1999), prior research using vertebrates cannot disentangle the linked effects of fluctuating temperatures and concordantly changing hydric environments on eggs, embryos, and offspring (Packard and Packard 1988). Despite considerable phylogenetic divergence, we compare below our findings with those obtained using lizards and snakes, due to the paucity of relevant studies on turtles (Georges et al. 1994; Doody 1999). Hatching success and days to pip Hatching success rates similar to, but slightly lower than, those found in this study were attained in studies of Apalone species in which incubation temperature stayed constant throughout incubation at some value between 25C and 30C (Ewert 1979; Packard et al. 1979, 1981; Janzen 1993b; Plummer et al. 1994). Doody (1999) found that hatching success for A. spinifera (LeSueur 1827) was slightly higher for eggs incubated at constant temperatures than for eggs incubated at fluctuating temperatures in artificial nests. These results could derive from extreme low temperatures experienced in the most shaded artificial nest in his study and may not be a property of fluctuating temperatures per se. Embryos subjected to higher daily fluctuation (30.5 4C) took substantially longer to pip (>2 days on average) than those at either the constant (30.5C) or lower fluctuation (30.5 2C) treatment (Table 1). A portion of this pattern may reflect differences among treatments in tendency of turtles to pip eggs earlier versus later in the day (a.m.: 59.0% from 30.5C, 56.7% from 30.5 2C, and 21.7% from 30.5 4C). Overall, this result is consistent with Shine et al. (1997), although both the mean and the variance of incubation temperature changed between treatments in their study, so disentangling these effects on incubation length is difficult and limits direct comparison with results of other studies. In contrast, greater daily temperature fluctuation reduced the incubation period in other squamate reptiles (Overall 1994; Shine and Harlow 1996); additional studies of various reptiles have detected no differential effect of constant and fluctuating incubation temperatures on incubation length (Georges et al. 1994; Andrews et al. 2000; Webb et al. 2001).

186 Constant-temperature studies of turtle embryos reveal that incubation length increases exponentially as incubation temperature decreases (Ewert 1979; Choo and Chou 1987; Leshem et al. 1991; Janzen 1993b; Ackerman 1994; Plummer et al. 1994; Doody 1999). Thus, the drop in incubation temperature to 26.5C for 12 h each day in our study may have disproportionately slowed embryonic development relative to the increased rate induced by the rise in incubation temperature to 34.5C for 12 h each day. In this vein, contrasting results on the phenotypic effects of constant versus fluctuating temperatures from studies of diverse taxa may be caused by intrinsic physiological limits (=developmental minima/maxima) rather than by phylogenetic differentiation. Body size Little variation was detected among body-size measures at hatching across temperature treatments. This result is largely consistent with findings from similar studies of reptiles (Shine et al. 1997; Doody 1999; Andrews et al. 2000). The exceptions are that tail length of a lizard increases (Shine and Harlow 1996) and that, in a snake, snout-vent length and relative tail length decrease and relative body mass increases (Webb et al. 2001) with increasing thermal variance; in another lizard, hatchling mass is greatest at intermediate thermal variance (Overall 1994). The explanation for these different results is unknown. Interestingly, however, the variance in all measures of body size in our study was lowest in the 30.5 4C environment compared to the other two, thus signifying slightly greater phenotypic uniformity (result not shown). Fluctuating temperatures during incubation in nature (Fig. 1) therefore might increase phenotypic uniformity within nests in this population. In support of this hypothesis, constant-temperature incubation studies (e.g., Janzen 1993b; Plummer et al. 1994) reveal a heightened variance in similar measures of body size when compared to the current experiment. In contrast to developmental rate, body size was most strongly affected by clutch even after accounting for initial egg mass (P 0.05 in all four cases). Strong clutch effects on size of neonatal reptiles are well known, so this result is no surprise (e.g., Shine and Harlow 1996; Shine et al. 1997; Andrews et al. 2000; Packard and Packard 2000; Webb et al. 2001). More unexpected is the lack of a clear temperature effect because: (1) length of incubation was influenced by temperature variance (Table 1), and (2) different constant temperatures elicit significant variation in body size (Janzen 1993b; Plummer et al. 1994). Performance Swimming speed of hatchlings from eggs incubated at a constant 30.5C was comparable to results from prior research on the same population (constant 30C in Janzen 1993b). Overall though, greater fluctuation in incubation temperature resulted in greater swimming speed (Table 1). This result accords with findings for running speed in hatchlings of a lizard (Shine and Harlow 1996). However, neither snakes (Shine et al. 1997; Webb et al. 2001) nor (apparently) A. spinifera (Doody 1999) exposed to greater thermal fluctuations as embryos swam faster as hatchlings. Increased thermal variance during development led to both faster swimming speed and a longer incubation period in this study. These two measures were also positively correlated (Table 2). Because turtles from the 30.5 4C treatment took longest to hatch on average, they may have had more time to mature and thus perform better in trial runs. In contrast, increased variance in incubation temperature led lizards to run faster after hatching, but was also linked to shorter incubation times (Shine and Harlow 1996). However, in light of our swimming-performance results, the interpretation of some findings from prior studies of A. mutica may need to be reconsidered. Because temperatures fluctuate continuously in natural nests (Fig. 1), hatchling A. mutica from past constanttemperature studies (e.g., Janzen 1993b) may actually have performed better if they been reared in the field. In other words, the potential for greater swimming speed was present, but constant incubation temperatures constrained its realization. Righting time in our study is a derivation of the rarely used measure of number of flips to fatigue (but see Doody 1999). While this latter test might measure the endurance of a hatchling, we did not use it because we questioned its ecological relevance. Survival of neonatal reptiles may depend on the first flip or even the first few flips, but little information related directly to survival ability in nature is likely to be revealed by flipping to fatigue. Rather, the speed with which an individual can right itself during a potential predation event seems to be a more ecologically relevant measure of antipredator ability (F.J. Janzen, personal observation), although other traits like burying behavior (Doody 1999) might be important as well. Regardless, righting time was not significantly influenced by clutch, temperature treatment, or the interaction between the two. Righting time did increase with an increasing variance in incubation temperature however (Table 1). Interestingly then, incubation conditions with increased thermal variance produced hatchlings that on average were faster swimmers and slower flippers, but these two variables were not significantly correlated across all turtles. That is, individual turtles that were faster swimmers were not necessarily slower flippers too (Table 2). Prior research on the influence of constant versus fluctuating incubation temperatures on performance variables other than swimming or running speed in neonatal reptiles once again reveals no clear pattern. At intermediate levels of thermal variance, basking time and overall activity levels were maximized in a lizard (Shine and Harlow 1996), whereas motivation (i.e., number of

investigator taps to instigate swimming) was minimized and endurance (i.e., number of times an animal surfaced) was maximized in a snake (Shine et al. 1997). Frequency of stopping during swimming trials fell and frequency of hiding during swimming trials rose with increased thermal variance in another snake (Webb et al. 2001). Burying speed was unaffected by thermal variance in A. spinifera (Doody 1999) but, interestingly, posthatching survival of a lizard in the field seemed to be enhanced by increased thermal variance during incubation (see Table 3 of Andrews et al. 2000). The variation in results among experiments again highlights the currently insufficient database for identifying possible generalities in phenotypic responses of reptilian neonates to thermal variance during embryonic development. These problems can be remedied readily by adopting a broadly comparative approach that minimizes methodological differences, another potential explanation for the lack of generalities currently observed. Even so, the insect literature reveals that generalities may be hard to come by (Ratte 1985). Our study is the first designed to examine the effects of both constant and controlled fluctuating temperatures on a wide array of traits in hatchling turtles. A number of experiments in reptiles have recognized the import of thermal variation in addition to mean temperature on embryonic sex determination (e.g., Valenzuela 2001). However, only Georges et al. (1994) directly explored the phenotypic effects of constant and fluctuating temperatures with identical means (on incubation time and sex ratio of hatchling sea turtles); Doody (1999), for example, sought to quantify constant temperature equivalents of fluctuating temperatures found in natural A. spinifera nests. Our results and those of other researchers suggest that, like our increased recognition of the importance of clutch and maternal effects (e.g., Packard and Packard 1993), thermal variance should continue to be accounted for explicitly in studies of embryonic and neonatal reptiles and other organisms. Indeed, future studies of phenotypic variation resulting from incubation-temperature differentiation should attempt to incorporate fluctuating temperature regimes. The resultant data may not only resemble natural variation more closely, but also might change our whole outlook on the biological causes and consequences of phenotypic variation (e.g., Shine and Elphick 2001). Acknowledgements We thank D. Robinson for assistance collecting eggs, J. Krenz and D. Willette for aid in constructing nest protectors, N. Valenzuela for the loan of temperature loggers, M. Knutzen for crucial help tending eggs in the laboratory, A. Bronikowski for statistical advice, and G. Packard, two anonymous reviewers, and members of the Janzen Laboratory for comments on the manuscript. Research was conducted under authority of scientific permit SC 14 0001 from the Iowa Department of Natural Resources. Turtles were handled in accordance with Iowa State University Animal Care protocol 1-0-4392-1-J. This project was supported in part by NSF grant DEB-9629529 to F.J.J. Journal paper no. J-19839 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa, project no. 3369, supported by the Hatch Act and State of Iowa Funds. References 187 Ackerman RA (1994) Temperature, time, and reptile egg water exchange. Israel J Zool 40:293 306 Andrews RM, Mathies T, Warner DA (2000) Effect of incubation temperature on morphology, growth, and survival of juvenile Sceloporus undulatus. Herpetol Monogr 14:420 431 Billerbeck JM, Schultz ET, Conover DO (2000) Adaptive variation in energy acquisition and allocation among latitudinal populations of the Atlantic silverside. Oecologia 122:210 219 Burggren WW, Just JJ (1992) Developmental changes in physiological systems. In: Feder ME, Burggren WW (eds) Environmental physiology of the amphibians. University of Chicago Press, Chicago, pp 467 530 Choo BL, Chou LM (1987) Effect of temperature on the incubation period and hatchability of Trionyx sinensis Wiegmann eggs. J Herpetol 21:230 232 Deeming DC, Ferguson MWJ (1991) Egg incubation: its effects on embryonic development in birds and reptiles. Cambridge University Press, New York Doody JS (1999) A test of the comparative influences of constant and fluctuating incubation temperatures on phenotypes of hatchling turtles. Chel Conserv Biol 3:529 531 Ernst CH, Barbour RW, Lovich JE (1994) Turtles of the United States and Canada. Smithsonian Institution Press, Washington, DC Ewert MA (1979) The embryo and its egg: development and natural history. In: Harless M, Morlock H (eds) Turtles: perspectives and research. Wiley, New York, pp 333 413 Ewert MA (1985) Embryology of turtles. In: Gans C, Billett F, Maderson PFA (eds) Biology of the reptilia, vol 14. Wiley, New York, pp 75 267 Fitch HS, Plummer MV (1975) A preliminary ecological study of the soft-shelled turtle Trionyx muticus in the Kansas River. Israel J Zool 24:28 42 Garland T Jr (1984) Physiological correlates of locomotory performance in a lizard: an allometric approach. Am J Physiol 247:R806 R815 Garland T Jr (1988) Genetic basis of activity metabolism. I. Inheritance of speed, stamina, and antipredator displays in the garter snake Thamnophis sirtalis. Evolution 42:335 350 Garland T Jr, Bennett AF (1990) Quantitative genetics of maximal oxygen consumption in a garter snake. Am J Physiol 259:R986 R992 Georges A, Limpus C, Stoutjesdijk R (1994) Hatchling sex in the marine turtle Caretta caretta is determined by proportion of development at a temperature, not daily duration of exposure. J Exp Zool 270:432 444 Hagstrum DW, Milliken GA (1991) Modeling differences in insect developmental times between constant and fluctuating temperatures. Ann Entomol Soc Am 84:369 379 Janzen FJ (1993a) An experimental analysis of natural selection on body size of hatchling turtles. Ecology 74:332 341 Janzen FJ (1993b) The influence of incubation temperature and family on eggs, embryos, and hatchlings of the smooth softshell turtle (Apalone mutica). Physiol Zool 66:349 373 Janzen FJ, Packard GC, Packard MJ, Boardman TJ, zumbrunnen JR (1990) Mobilization of lipid and protein by embryonic snapping turtles in wet and dry environments. J Exp Zool 255:155 162 Leshem A, Dmi el R (1986) Water loss from Trionyx triunguis eggs incubating in natural nests. Herpetol J 1:115 117 Leshem A, Ar A, Ackerman RA (1991) Growth, water, and energy metabolism of the soft-shelled turtle (Trionyx triunguis) embryo: effects of temperature. Physiol Zool 64:568 594 Miller K, Packard GC, Packard MJ (1987) Hydric conditions during incubation influence locomotor performance of hatchling snapping turtles. J Exp Biol 127:401 412 Overall KL (1994) Lizard egg environments. In: Vitt LJ, Pianka ER (eds) Lizard ecology: historical and experimental perspectives. Princeton University Press, Princeton, pp 51 72

188 Packard GC, Packard MJ (1988) Physiological ecology of reptilian eggs and embryos. In: Gans C, Huey RB (eds) Biology of the reptilia, vol 16. Liss, New York, pp 523 605 Packard GC, Packard MJ (1993) Sources of variation in laboratory measurements of water relations of reptilian eggs and embryos. Physiol Zool 66:115 127 Packard GC, Packard MJ (2000) Developmental plasticity in painted turtles, Chrysemys picta. Funct Ecol 14:474 483 Packard GC, Taigen TL, Boardman TJ, Packard MJ, Tracy CR (1979) Changes in mass of softshell turtle (Trionyx spiniferus) eggs incubated on substrates differing in water potential. Herpetologica 35:78 86 Packard GC, Taigen TL, Packard MJ, Boardman TJ (1981) Changes in mass of eggs of softshell turtles (Trionyx spiniferus) incubated under hydric conditions simulating those of natural nests. J Zool 193A:81 90 Packard MJ, Packard GC (1991) Sources of calcium, magnesium, and phosphorus for embryonic softshell turtles (Trionyx spiniferus). J Exp Zool 258:151 157 Pennisi E (2002) Good diet hides genetic mutations. Science 296:1011 Plummer MV, Shadrix CE, Cox RC (1994) Thermal limits of incubation in embryos of softshell turtles (Apalone mutica). Chel Conserv Biol 1:141 144 Ratte H (1985) Temperature and insect development. In: Hoffman KH (ed) Environmental physiology and biochemistry of insects. Springer, Berlin Heidelberg New York, pp 33 66 Schrag SJ, Ndifon GT, Read AF (1994) Temperature-determined outcrossing ability in wild populations of a simultaneous hermaphrodite snail. Ecology 75:2066 2077 Shine R, Elphick MJ (2001) The effect of short-term weather fluctuations on temperatures inside lizard nests, and on the phenotypic traits of hatchling lizards. Biol J Linn Soc 72:555 565 Shine R, Harlow PS (1996) Maternal manipulation of offspring phenotypes via nest-site selection in an oviparous lizard. Ecology 77:1808 1817 Shine R, Madsen TRL, Elphick MJ, Harlow PS (1997) The influence of nest temperatures and maternal brooding on hatchling phenotypes in water pythons. Ecology 78:1713 1721 Valenzuela N (2001) Constant, shift, and natural temperature effects on sex determination in Podocnemis expansa turtles. Ecology 82:3010 3024 Webb JK, Brown GP, Shine R (2001) Body size, locomotor speed and antipredator behaviour in a tropical snake (Tropidonophis mairii, Colubridae): the influence of incubation environments and genetic factors. Funct Ecol 15:561 568 Wyneken J, Salmon M (1992) Frenzy and postfrenzy swimming activity in loggerhead, green, and leatherback hatchling sea turtles. Copeia 1992:478 484