Effects of Incubation Temperature on Growth and Performance of the Veiled Chameleon (Chamaeleo calyptratus)

Transcription

1 JOURNAL OF EXPERIMENTAL ZOOLOGY 309A: (2008) A Journal of Integrative Biology Effects of Incubation Temperature on Growth and Performance of the Veiled Chameleon (Chamaeleo calyptratus) ROBIN M. ANDREWS Department of Biological Sciences, Virginia Polytechnic Institute and State University, Blacksburg, Virginia ABSTRACT I evaluated the effect of incubation temperature on phenotypes of the veiled chameleon, Chamaeleo calyptratus. I chose this species for study because its large clutch size (30 40 eggs or more) allows replication within clutches both within and among experimental treatments. The major research objectives were (1) to assess the effect of constant low, moderate, and high temperatures on embryonic development, (2) to determine whether the best incubation temperature for embryonic development also produced the best hatchlings, and (3) to determine how a change in incubation temperature during mid-development would affect phenotype. To meet these objectives, I established five experimental temperature regimes and determined egg survival and incubation length and measured body size and shape, selected body temperatures, and locomotory performance of lizards at regular intervals from hatching to 90 d, or just before sexual maturity. Incubation temperature affected the length of incubation, egg survival, and body mass, but did not affect sprint speed or selected body temperature although selected body temperature affected growth in mass independently of treatment and clutch. Incubation at moderate temperatures provided the best conditions for both embryonic and post-hatching development. The highest incubation temperatures were disruptive to development; eggs had high mortality, developmental rate was low, and hatchlings grew slowly. Changes in temperature during incubation increased the amongclutch variance in incubation length relative to that of constant temperature treatments. J. Exp. Zool. 309A: , r 2008 Wiley-Liss, Inc. How to cite this article: Andrews RM Effects of incubation temperature on growth and performance of the veiled chameleon (Chamaeleo calyptratus). 309A: For reptiles, temperatures experienced by embryos during incubation affect the phenotype after hatching or birth. The phenotypic attributes affected include body size and shape, survival, running speed, temperature preference, growth rate, antipredator behaviors, and the sex of some turtles, some lizards, and all crocodilians (Burger, 91; Janzen and Paukstis, 91; Downes and Shine, 95; Shine et al., 97; Deeming, 2004; Booth, 2006). For example, eggs of wall lizards (Podarcis muralis) incubated at 321C produced hatchlings with significantly shorter body lengths, and shorter tails, heads, and femurs relative to body length, than those incubated at 26 and 291C (Braña and Ji, 2000). Eggs of Sceloporus virgatus incubated at C (diel temperature fluctuation) produced hatchlings that grew more rapidly, ran more slowly, and selected lower body temperatures than hatchlings from eggs incubated at C (Qualls and Andrews, 99). More examples of temperature effects on hatchling phenotypes are provided in recent reviews (Rhen and Lang, 2004; Booth, 2006). The assumption of these studies is, of course, that at least some environmentally induced variation in the phenotypes of early life stages reflects variation in ultimate fitness. Grant sponsor: Morris Animal Foundation; Grant number: D01ZO-79. Correspondence to: Robin M. Andrews, Department of Biological Sciences, Virginia Tech, Blacksburg, VA randrews@vt.edu Received 26 November 2007; Revised 14 March 2008; Accepted 23 April 2008 Published online 30 May 2008 in Wiley InterScience (www. interscience.wiley.com). DOI: /jez.470 r 2008 WILEY-LISS, INC.

2 436 ANDREWS Evaluating the extent to which this is true is challenging because of the obvious difficulties in determining the reproductive success of individuals and their descendents. Despite the logistic difficulties in determining how and whether the temperature experienced by embryos during incubation affect individual fitness, a few comparatively long-term studies have now been conducted. These studies indicate that while environmentally induced phenotypes can persist, they often do not, and traits that persistent over time in one study may not in another. For example, morphological traits of Sceloporus undulatus individuals that were present at hatching persisted over 5 7 months after release in the field (Andrews et al., 2000). In contrast, morphological traits of Bassiana duperreyi that differed at hatching disappeared early in the observation period while a performance trait (running speed) differed between temperature treatments at hatching and 5 months later (Elphick and Shine, 98). Similarly, running speed of Gallotia galloti individuals associated with incubation temperature persisted over 10 months (Vanhooydonck et al., 2001). Finally, Qualls and Shine (2000) found that morphological and behavioral phenotypes of Lapropholis guichenoti at the end of months of observations under semi-natural conditions were largely the result of the rearing environment of hatchlings; effects of incubation temperature were inconsequential once the effects of the time of hatching were removed. Phenotypic variation associated with incubation temperature thus has the potential, at least, to affect fitness. To provide experimental consistency, the great majority of studies on the effects of incubation temperature on phenotype use constant temperature regimes during incubation. Although Andrews et al. (2000) did not find any differences between incubation length and phenotypes of hatchlings from eggs incubated at a constant 281C and from a temperature regime that averaged 281C but fluctuated C during the diel cycle, a number of observations suggest that the temporal pattern of temperature variation during development may affect phenotypes. The sex of reptiles with temperature-dependent sex determination is determined only during the period of organogenesis when gonads differentiate (Janzen and Paukstis, 91). Quantitative traits such as size or locomotory capabilities may also be sensitive to temperature at particular stages. Another observation is that the rate of development is positively related to temperature at early stages, but largely independent of temperature later in development (Birchard and Reiber, 95; Andrews, 2004). Finally, natural variation in incubation temperature is associated with phenotypes of hatchlings and exposure to high temperature early in development affects phenotypes of hatchlings more than later in development (Shine and Elphick, 2001). Accordingly, the processes of differentiation, which occur early in development, may be more affected by changes in temperature than the processes by which embryos increase dramatically in mass late in development. To evaluate the effect of temperature variation during incubation on lizard phenotypes, I made observations on the veiled chameleon, Chamaeleo calyptratus. I chose this species for study because it is widely available in the pet trade, both exported directly from the Middle East (Yemen) and from captive breeding, relatively hardy, and has a large clutch size (30 40 eggs or more in captivity) that allows replication within clutches both within and among experimental treatments. Sex is determined genetically (Andrews, 2005). Because eggs are laid when embryos are gastrulae, incubation temperature can be controlled during the almost entire period of development. The research had three major objectives. The first was to assess the effect of incubation temperature per se on phenotype. I therefore incubated chameleon eggs at relatively low, moderate, and high temperatures (25, 28, and 301C, respectively) throughout the incubation period. The second objective was to determine whether the best incubation temperature for development also produced the best hatchlings. The third objective was to determine whether phenotypes are affected by the temporal pattern of temperature exposure during development. I therefore exposed eggs incubated at 281C to relatively low (251C) or high (301C) incubation temperatures at the time when organogenesis is complete and the embryo enters the growth phase of development and made two alternative predictions. The critical period prediction is that embryos should be more sensitive to incubation temperature during organogenesis, the period of tissue and organ formation, than later in development when an increase in size is the dominant developmental process (Andrews, 2004). According to this prediction, phenotypes of individuals incubated at the same temperature during organogenesis should be more similar to one another than individuals incubated at different temperatures. The duration of exposure prediction is that phenotypes of individuals

3 INCUBATION TEMPERATURE AND CHAMELEON PHENOTYPE 437 incubated at the same temperature during the growth phase of development, which takes places over a substantially longer period than organogenesis, should be more similar to one another than individuals incubated at different temperatures. To test these predictions, I determined egg survival and incubation length and measured body size and shape, selected body temperatures, and locomotory performance of lizards after hatching at regular intervals from hatching to 90 d, or just before sexual maturity. MATERIALS AND METHODS Source of eggs and general husbandry of eggs and lizards Observations were made on five clutches laid between 3 and 25 April 2004 in a breeding colony in my laboratory. Eggs from each clutch were weighed and numbered sequentially with a fine India ink pen within 24 hr of oviposition and placed in a plastic shoe box container partially filled with moistened incubation media. Eggs were buried leaving roughly one-third of their top surface exposed so that they could be monitored without disturbance. Containers were placed in a Percival environmental chamber at a constant 281C; some eggs from each clutch were shifted later to other temperature treatments according to experimental protocols (see below). Temperature within an egg container in each of the three environmental chambers was recorded daily; temperatures were adjusted if necessary to maintain the targeted temperature. Mean temperatures for the three environmental chambers averaged 24.8, 28.0, and 29.91C during incubation. Containers were rotated within each chamber several times a week to minimize the effect of temperature gradients within the chamber on development. Of the initial 234 eggs in the five clutches, 201 were used in the experiments described here. Of the remaining 33 eggs, about half were sampled to determine the embryo stage and about half died before the time when eggs were allocated to experimental treatments. Eggs were incubated in moistened ecopeat (High Sierra Exotics, Quincy, CA) at a water potential of 280 kpa (150 g H 2 O per 100 g ecopeat) based on a standard curve established by vapor pressure psychometry that related water content to water potential (Andrews, unpublished data). Water was added to the containers once or twice a week to return the water content to its original value. We did not vary moisture during incubation because both field and laboratory studies indicate that the effects of temperature far outweigh the effects of moisture on phenotypes of individuals pre- and post-hatching (Flatt et al., 2001; Warner and Andrews, 2002). Containers were checked daily once hatching started. At hatching, lizards were identified individually with numbers written on their sides with a fine Sharpie marker. They were assigned sequential numbers to avoid associating individuals with either their clutch or treatment. Cages were checked two three times a day and lizards were renumbered when they shed. On four occasions, two individuals shed and could not be distinguished (e.g. two unnumbered individuals of the same size and sex in the same cage); these eight individuals were removed from the experiment. Five other individuals died as the result of various accidents. These problems involved individuals in four of the five clutches and all treatments; given the small numbers, experimental bias is unlikely. Hatchlings were initially housed in one of four glass-sided terraria (36H 75W 32D cm). Hatchlings were added to a terrarium as they hatched until it contained a group of 20 individuals. Because hatching occurred in the same treatment order in all clutches, groups contained representatives of four or (usually) five clutches. After 2 4 weeks hatchlings were moved to screened cages (77H 61W 41D cm); the group size was decreased as they grew such that by 3 months of age they were housed in groups of 3 6. Individuals were also shifted between groups in terraria or screened cages as necessary to maintain sets of individuals of similar size. Juveniles lived together amicably; only as they approached sexual maturity at about 4 months did individuals become aggressive toward one another. Terraria and cages were furnished with a dense network of branches and plastic plants for climbing and shelter. General lighting was provided by windows and by ultraviolet-b-emitting fluorescent bulbs hung immediately over cages ( hr). Basking opportunities were provided by watt incandescent lamps ( hr) hung at one end of the terraria or at the top of screened cages. Terraria were misted with water two or three times a day, and a drip system for screened cages provided a stream of water droplets on foliage for min a day. Hatchlings were fed crickets ad libitum once or twice daily. Crickets were dusted at each feeding with commercial dietary supplements (Nutrition Support Services Inc.:

4 438 ANDREWS Quantum Series Lowland Chameleon Dust, Pembroke, VA; RepCal Calcium with Vitamin D3 and RepCal Herptivite, Los Gatos, CA) to provide a balanced intake of nutrients. Crickets had a continuous supply of greens and carrots in addition to cricket chow to indirectly provide a balance of nutrients to the lizards. Staging, experimental design, and sampling protocols The times when treatments were initiated and embryos were sampled were based on developmental studies of C. calyptratus embryos at 281C (Andrews, 2004; Andrews and Donoghue, 2004). Embryos were staged using criteria of Dufaure and Hubert ( 61) as modified for C. calyptratus (Andrews, 2007). Embryos are in diapause at oviposition and development does not resume for d (Andrews and Donoghue, 2004). Although development is not initiated at the same time for all clutches, development is synchronous within clutches such that by stage 37 embryos differ by no more than one stage from other eggs from the same clutch sampled at the same time (Andrews, unpublished data). Neurulation is completed in about 8 d and encompasses stages Organogenesis, differentiation of tissues and organs (muscles, gut, heart, gonads, limbs, etc.), is completed in about 30 d and encompasses stages The final 80 d of development is characterized by a substantial increase in mass and encompasses stage 35 to hatching at stage 40. The three incubation temperatures used in experiments were 25, 28, and 301C. I predicted that the best incubation temperature would be at 281C because incubation at 251C would prolong development relative to 281C, and 301C is likely stressful because it is at the upper end of the constant temperature range for successful incubation (Necas, 99; Schmidt, 2001; Andrews, 2007). These temperatures fall within the range of nest temperatures in nature for Chamaeleo chamaeleon, a close relative of C. calyptratus. The mean nest temperature for C. chamaeleon during spring and summer is about 251C and in August, just before hatching, the mean nest temperature is about 301C (Díaz-Paniagua, 2007; Andrews et al., 2008). Incubation at a constant temperature is appropriate for Chamaeleo species because females place their nests well below the soil surface where diel variation in temperature is substantially lower than seasonal variation. For example, the diel range in temperature in the cm deep nests of C. chamaeleon is no more than 1 21C at any time during incubation. In contrast, the monthly mean temperature from the end of developmental arrest in the spring through hatching in the fall ranges from 20 to 301C (Díaz- Paniagua, 2007; Andrews et al., 2008). Eggs were incubated at 281C until 70 d when one egg per clutch was sampled. Because all sampled embryos had resumed development (Four were neurulae and one was stage 22), sub-sets of eggs from each clutch were assigned to the five experimental treatments at this time (Fig. 1). These treatments were (1) a control, eggs continued to be incubated at 281C with no change in temperature during incubation, (2) change to 251C for the remainder of incubation, (3) change to 301C for the remainder of incubation, (4) no change in temperature until embryos reached stage 35, then a change to 251C, and (5) no change in temperature until embryos reached stage 35, then a change to 301C. Treatments were designated at 28/28, 25/25, 30/30, 28/25, and 28/30, respectively, with the first number representing the temperature from the initiation of the experiment at 70 d to stage 34 (organogenesis) and the second, the temperature from stage 35 to hatching (growth in mass). At day 100, one egg per clutch was sampled from eggs allocated to treatments 4 and 5. Embryos in three of the clutches were at stage 34.5 or 35; eggs from these clutches were shifted to the 25 and 301C chambers, as appropriate. Embryos from two clutches were at stage 33; eggs from these clutches were shifted 4 5 d later given that each stage of organogenesis lasts about 2 d. When eggs were Fig. 1. Temperatures experienced by eggs of Chamaeleo calyptratus during incubation. Eggs were incubated at 281C during the initial 70 d when embryos were diapausing gastrulae. Incubation temperatures were reduced to 251C or increased to 301C at 70 or at 100 or 105 d as indicated. Treatment (Trt.) designations reflect temperatures during 70 to 100 d/100 (105) d to hatching. Ov. indicates oviposition at day 0 and H indicates hatching at day 180.

5 INCUBATION TEMPERATURE AND CHAMELEON PHENOTYPE 439 staged, the mass of the egg was recorded, and the embryo was preserved in alcohol. Effects of incubation temperature Effects of incubation temperature on development were assessed from observations on survival to hatching, length of incubation (oviposition to hatching), mass at hatching (0 d) and at 30, 60, and 90 d, and morphological measurements at 3 5, 30, 60, and 90 d. Measurements used to assess body shape were snout vent length (SVL), cask length, head length, head width, femur length, and tail length. The SVL was the distance from the tip of the snout to the vent, the cask length was the distance from the rear edge of the cask at mid-line to the tip of the snout, the head length was the distance from the most posterior extent of the mouth opening to the tip of the snout, the head width was the distance across the head between the most posterior extents of the mouth, the femur length was the distance from the insertion of the femur with the body to the maximum extent of the knee with the chameleon positioned such that its right femur was parallel to its body and the knee bent at 901, and the tail length was the distance between the vent and the tip of the tail. The SVL and the tail length were measured with a ruler and other measurements were made with a dial caliper. To assess locomotory performance, chameleons were sprinted on a race-track consisting of a rod suspended vertically from the center of a rectangular frame. Rods were 2.0 mm in diameter for hatchlings and 2.9 mm in diameter for older individuals. The frame s top was covered with plastic vines to provide apparent shelter to the lizard. Chameleons in nature use camouflage as a general protection from predators but flee rapidly toward dense vegetation when a perceived predator approaches (Cuadrado et al., 2001; Andrews, personal observation). Sprint speed thus has biological relevance for chameleons. Sprint speed was measured at 3 5, 30, and 60 d. Individuals were equilibrated at one of two ambient temperatures, 25 and 321C for min before trials. The temperature of the initial trial was alternated daily effectively randomizing the order in which individuals in each age group experienced the 25 and 321C trials. Individuals were released on the lower end of the rod at the beginning of a set of three trials at each temperature. Trial 1 was an orientation trial: when the lizard was released, the observer moved behind a blind and the lizard was observed for 4 min or until it climbed to the top of the rod. The lizard was then moved into an opaque cup with plastic vegetation until other lizards had finished their orientation trials. For the second and the third trials, each lizard was placed on the bottom of the rod and was encouraged to sprint by gently touching its tail to keep it moving. When it reached the top it was again placed in the cup until other lizards finished their trials. Lizards had 3 5 min to rest between sprint trials. The time required for individuals to climb between two marked positions on the rod (42 cm for hatchlings, 63 cm for individuals aged 30 and 60 d) was recorded using a stopwatch. When trials were completed at one temperature, the temperature was changed (20 30 min), and the three trials were conducted at the second temperature. The shorter of the times recorded for the two sprint trials was used as the measure of speed at each temperature. Selected body temperatures ðt sel Þ were measured in the lizard s home cages between 1,100 and 1,400 hr at regular intervals from hatching to 60 d of age. During the observation period, the lowest ambient temperatures in the cages were C and the warmest were C. The ambient temperature varied linearly from 25 to 351C at a rate of 0.41C/cm. Body temperatures were measured with an infrared thermometer (Raytek Raynger ST60, Santa Cruz, CA) to minimize disturbance to the lizards. For calibration, readings from the infrared thermometer were compared with simultaneous readings of body (cloacal) temperature taken with a thermocouple thermometer. At equilibrium, body temperatures recorded by the infrared thermometer were within a few tenths of a degree of body (cloacal) temperatures of live or freshly killed lizards. Body temperatures were recorded with the thermometer held perpendicular to the trunk with all laser sights located well within the trunk area (Hare et al., 2007). Mean T sel were determined for 95 individuals with the modal number of four individuals (range 5 2 5) in each clutch/treatment combination. Means for 91 individuals were based on observations, one individual had six observations, one had eight and two had nine. Data for individuals that were culled at 30 d (see below) were excluded from analyses because they had relatively few observations of selected body temperature. Protocols for culling The roughly 200 hatchlings that were produced exceeded available caging once they reached 2 4 g in mass and had to be housed in small groups.

6 440 ANDREWS Therefore, after the first set of observations at hatching, individuals were randomly culled such that each clutch/treatment combination was reduced to two females and two males. Observations were made on these individuals through 90 d of age. Statistical analyses Preliminary analyses incorporated temperature treatment, clutch, age, and sex as class variables. Interactions between age and treatment and between age and clutch were significant for most dependent variables. Sex was not significant either as a class variable or in interaction terms in any model. Therefore, subsequent analyses were run separately for each age group and sex was not considered further as a class variable. Temperature was a fixed factor and clutch was a random factor in mixed model analyses of variance (ANOVAs) (Sokal and Rohlf, 81). Accordingly, temperature effects were tested with the interaction mean square rather than error mean square. Residual analyses were used to adjust independent variables for confounding factors: residuals were determined for hatchling mass as a function of initial egg mass, mass at 30, 60, and 90 d as a function of initial hatchling mass and T sel, linear measures of head, limb, and tail dimensions as a function of the SVL, and sprint speed as a function of the SVL and the femur length. These adjustments were made because initial egg mass affects hatchling mass independently of clutch identity, body temperature affects growth rate, size per se affects linear dimensions, and size and relative limb length affect locomotory performance (Sinervo and Adolph, 94; Elphick and Shine, 98; Braña and Ji, 2000; Vanhooydonck and Van Damme, 2001). Parametric analyses were conducted with SAS software (SAS, 97). Least squares means procedures were used for a posteriori tests. The significance was at Po0.05 throughout. highest survival and the two with the lowest survival, cells were combined because of low expected frequencies) but did vary among treatments (w , d.f. 5 1, Po0.01). The three coolest treatments (25/25, 28/25, 28/28) had higher survival than the two warmest treatments (28/30, 30/30) with 96 and 86% survival, respectively (cells were combined because of low expected frequencies). The incubation length varied among treatments and among clutches (Fig. 2) (Treatment: F 4, , Po0.001; Clutch: F 4, , Po0.001). Because of a clutch by treatment interaction (F 16, , Po0.001), however, independent contrasts were made within clutches and within treatments using one-way ANOVAs. The incubation length varied among treatments within all clutches (Pso0.003). The incubation length also varied among clutches within treatments except the 28/28 treatment (Table 1). For the three coolest treatments, the incubation length declined as average temperature increased, but for the two warmest treatments, clutch identity interacted with treatment effects. For two clutches, incubation was substantially longer in the 28/30 and 30/30 treatments than the 28/28 RESULTS Egg mass, embryo survival, and incubation length Clutch means of egg mass at oviposition ranged from 1.04 to 1.24 g (overall mean g). Egg survival was high with 92% of eggs producing viable hatchlings. Egg survival did not vary among clutches (w , d.f. 5 1, P40.05, contrasts of survival between the three clutches with the Fig. 2. Total length of incubation for eggs from five clutches of C. calyptratus. Eggs were incubated under five temperature regimes (see Fig. 1). Respective number of eggs for the 25/25, 28/25, 28/28. 28/30, and 30/30 treatments were 6, 9, 5, 7, and 8 clutch C5 eggs, 19, 5, 5, 4, 16 clutch D4 eggs, 12, 4, 4, 3, 9 clutch E7 eggs, 14, 4, 4, 4, and 16 clutch S3 eggs, and 10, 3, 4. 3, 10 clutch T6 eggs. Error bars represent standard deviations, and means are displaced for clarity. Results of statistical analyses (clutch effects) are presented in Table 1.

7 INCUBATION TEMPERATURE AND CHAMELEON PHENOTYPE 441 treatment. For the other three clutches, incubation in the 28/30 and 30/30 treatments took less time or was comparable to the 28/28 treatment. Selected body temperatures Mean T sel of individuals ranged from 25.1 to 31.91C. Individuals with higher T sel had larger body sizes (30 d: r , P , n 5 94; 60 d: r , P , n 5 93; 90 d: r , P , n 5 89, Spearman rank correlations). T sel, however, was not related to either treatment or clutch (overall F 24, , P , twofactor ANOVA). The body temperature of hatchling lizards is directly related to their food intake and hence growth (Sinervo and Adolph, 94). Hence, the strong positive correlation between body mass (i.e. growth) and T sel independent of treatment or clutch identity suggests that individuals had differential access to thermal resources. Although I did not observe overtly aggressive behavior among cage mates and cage mates were matched by size, subtle behavioral interactions could have facilitated or impaired thermoregulation by certain individuals. TABLE 1. Results of one-way ANOVAs for clutch effects on incubation length by treatment (see Fig. 2) and associated variance components Treatment Source d.f. MS F- ratio P s 2 c % s 2 c 25/25 Clutch o Error /25 Clutch o Error /28 Clutch Error /30 Clutch Error /30 Clutch 4 1, Error Error is the variance within clutches, s 2 c is the variance among clutches, and % s 2 c is the percent of the total variance attributed to among-clutch effects (Sokal and Rohlf, 81, Box 9.2). ANOVA, analysis of variance. Body size and body shape Body size varied as a function of incubation temperature (Table 2, Fig. 3). At hatching (day 0), individuals from the three coolest treatments were heavier than individuals from the two warmest treatments. At 60 d, however, the largest individuals were associated with intermediate (28/28 and 28/30) temperature treatments. At 90 d, treatment effects were not significant but the pattern of size variation was the same as at 60 d. Inspection of clutch means (Fig. 3) suggests that clutch treatment interactions likely overrode treatment effects at 90 d. At both 60 and 90 d, however, the clutch treatment interaction involved two clutches (D4 and T6) that had their highest means in the 28/28 treatment and three clutches (C5, E7, and S3) that had their highest means in the 28/30 treatment. The incubation temperature was only weakly related to shape dimensions (Table 3). Overall models were significant in only six of 20 possible age-dimension comparisons. Of these six, five clutch contrasts were significant and three treatment contrasts were significant. The cask length decreased with increasing incubation temperature at 0 d, the femur length was longest in the 28/28 treatment at 0 d and increased with incubation temperature at 60 d (Fig. 4). Given, however, that none of these treatment effects would be significant if significance levels were corrected for the number of comparisons, I consider that the results of shape analyses may be spurious. Sprint speed Sprint speed was affected by trial temperature, SVL, and femur length but not treatment or clutch. Chameleons ran slower at 25 than at 321C at all ages (Pso0.001, paired t-tests, t s , 14.2, and 10.7 at 0, 30, and 60 d). Mean sprint speeds at 25 and 321C were 2.0 and 2.6 cm/s at 3 5 d (n 5 176), 4.2 and 5.4 cm/s at 30 d (n 5 98), and 5.6 and 7.0 cm/s at 60 d (n 5 93), respectively. Sprint speed increased as a function of SVL in all TABLE 2. Results of two-factor mixed model ANOVAs for body mass (g) at 0, 60, and 90 days of age Age (d) Treatment Clutch Interaction 0 F 4, , Po0.001 F 4, , Po0.001 F 16, , P F 4, , Po0.05 F 4, , P F 16, , P F 4, , P40.05 F 4, , Po0.001 F 16, , P For observations at day 0, masses were represented by residuals of the regression of hatchling mass on the initial egg mass. For observations at 30, 60, and 90 d, masses were represented by residuals of the regression of mass on initial hatchling mass and T sel (see text for details). Overall models were significant at Po0.002 except at 30 d when P (results not shown). ANOVA, analysis of variance.

8 442 ANDREWS temperature and clutch effects. Overall models were significant in only two of six analyses (25 and 321C trials at 60 d). In those comparisons, only the clutch (F 4, , P and F 4, , P , respectively) was significant, whereas treatments did not differ (F 4, , PZ0.5 and F 4, , P40.05, respectively). DISCUSSION Fig. 3. Clutch and overall means for body mass of hatchling C. calpytratus at 0, 60, and 90 d. Clutch means are indicated by small symbols connected by lines (see Fig. 2 for key) and treatment means by large open circles. Means with the same lower case letter do not differ (P40.05). Treatment effects were not significant at 90 d and treatment contrasts are not shown. Results of statistical analyses are presented in Table 2. six age temperature combinations (three ages and two temperatures) and sprint speed increased as a function of femur length in four of the six (Pso0.05, regression analyses). In multiple regression analyses with SVL and femur length as independent variables, four of six overall models were significant, and in these analyses speed was explained by SVL in two of the four (Pso0.01) and femur length did not explain any additional variation in speed (all Ps40.05). Residuals of speed on SVL were therefore used to assess Recent reviews emphasize that although incubation temperature affects a wide range of phenotypic traits, effects are not consistently detectable, can be time dependent, indirect, or transitory (Deeming, 2004; Rhen and Lang, 2004). I also found a diversity of treatment effects. The incubation temperature affected the length of development and egg survival. After hatching, the incubation temperature affected therateofgrowthandsomecomponentsofshape, but had no effect on sprint speed and selected body temperature. Selected body temperature, however, had an indirect affect on body mass (growth) independently of treatment and clutch. I also found temporal effects of incubation temperature, and effects that were modified by clutch identity. I will discuss these results first with regard to the predictions that the 28/28 treatment should provide the best conditions for both embryonic and posthatching development and second with regard to the prediction that a temporal change in incubation temperature will alter phenotypes relative to those of embryos and hatchlings incubated at constant temperature throughout incubation. Finally, I comment on the value of assessing clutch effects. An assumption of this and other studies is that particular phenotypic traits are better than others in the sense that they are likely to improve individual fitness. Many such associations between phenotype and fitness make biological sense and thus provide reasonable first approximation for evaluating the fitness consequence of manipulations of a phenotype in a laboratory setting (Shine and Elphick, 2001). For example, I assume that high survival is better than low survival, shorter incubation periods are better than longer ones because early-hatching individuals have a headstart on accruing resources, large body size of individuals before maturity is better than small body size because of a greater ability to capture prey, escape predators, etc., fast growth is better than slow growth because individuals will reach sexual maturity sooner, and fast running speeds are better than slow ones to escape from predators, capture food, etc. (Packard and Packard, 88).

9 INCUBATION TEMPERATURE AND CHAMELEON PHENOTYPE 443 TABLE 3. Results of two-factor mixed model ANOVAs for shape variables Days Treatment Clutch Cask length 0 F 4, , Po0.01 F 4, , P Head length 0 F 4, , P40.05 F 4, , P F 4, , P40.05 F 4, , P Femur 0 F 4, , Po0.01 F 4, , Po F 4, , Po0.02 F 4, , P Tail length 0 F 4, , P40.05 F 4, , Po0.001 Observations were represented by residuals of regressions of morphological dimensions on the SVL at each age. Results are given only when overall models were significant (Po0.05). Treatment means for the three significant shape dimensions are shown in Figure 4. SVL, snout vent length; ANOVA, analysis of variance. Fig. 4. Overall treatment means for the significant shape variables of C. calyptratus hatchlings. Means with the same lower case letter do not differ (P40.05). Results of statistical analyses are presented in Table 3. Effect of incubation temperature per se on development pre- and post-hatching The prediction that the 28/28 treatment would provide the better incubation conditions than the 25/25 and 30/30 treatments for embryonic and post-hatching development was born out by the aggregate of observations on incubation length, egg survival, hatchling mass, and growth after hatching. The incubation length of reptiles decreases (more rapid embryonic development) as the incubation temperature increases until some minimal value is reached (Muth, 80). A subsequent increase in incubation length (and embryo mortality) at even higher temperatures is presumably because of heat-related disruption of developmental processes (Packard and Packard, 88; Andrews, 2007). The incubation length (Fig. 1) and survival of C. calyptratus eggs followed this general pattern. The mean incubation length was longest in the 25/25 treatment, shortest in the 28/ 28 treatment, and, depending on clutch, similar in the 30/30 and 28/28 treatments or longer in the 30/ 30 than the 28/28 treatment. Egg survival and hatchling mass were higher in the cooler 25/25, 28/ 25, and 28/28 treatments than in the warmer 28/ 30 and 30/30 treatments. The combination of a relatively short incubation length, relatively high survival, and relatively large size at hatching in the 28/28 treatment supports the prediction that 281C is the best temperature for embryonic development of C. calyptratus. In addition, by 60 d after hatching, individuals from the 28/28 treatment exhibited the highest growth rates of the three constant temperature treatments (Fig. 3). The growth advantage of individuals from the 28/28 treatment persisted at least through 90 d although differences among treatments were not statistically significant at this time. Incubation at both relatively low and relatively high temperatures reduced the apparent fitness of

10 444 ANDREWS hatchlings. For the 25/25 treatment, low developmental rates delayed hatching for almost 1 month relative to the 28/28 treatment. Although individuals in the 25/25 treatment had relatively high survival and relatively large size at hatching, subsequent low growth rates eroded any attendant advantage. For the 30/30 treatment, embryonic development in some clutches, at least, was slow, egg survival was low, and individuals had low growth rates after hatching. In addition, high temperature per se appeared to disrupt development as judged by high variability in the incubation length both within and among clutches for eggs in the 30/30 treatment (Table 2). The longterm consequences of incubation temperatures that are physiologically stressful to embryos are unknown, but the immediate outcome of prolonged development and low growth rates after hatching would be a reduction in fitness because individuals would reach sexual maturity substantially later than individuals from clutches incubated at moderate temperatures. Effect of temporal pattern of temperature exposure on phenotype Phenotypes associated with the treatments in which temperature was increased or decreased at the end of organogenesis (28/25 and 28/30) did not match the predictions of either the critical period or duration of exposure hypothesis. The critical period hypothesis was rejected because no set of phenotypes was associated with incubation at 281C during the organogenesis phase of development (28/25, 28/28, and 28/30 treatments). The duration of exposure hypothesis was rejected because phenotypes from eggs incubated at 251C (25/25 and 28/25 treatments) and at 301C during the growth phase (28/30 and 30/30 treatments) did not group accordingly. Instead, phenotypes associated with the 28/25 and 28/30 treatments tended to be intermediate between adjacent constant temperature regimes thus reflecting an averaging of temperature during incubation across developmental phases. Nonetheless, the among-clutch variance in incubation length associated with the 28/25 and 28/30 treatments was much greater than that of adjacent constant temperature treatments (Table 1). The 25/25, 28/25, and 28/28 treatments had comparable within-clutch variances but the variance among clutches for the 28/25 treatment was higher than for the 25/25 and 28/28 treatments. Similarly, the 28/30 and the 30/30 treatments had comparable (but relatively high) within-clutch variances, but the 28/30 treatment had a substantially higher among-clutch variance than the 30/30 treatment. These observations indicate that a change in incubation temperature at the end of organogenesis had clutch-specific effects on the rate of embryonic development. The shift in temperature of the 28/30 treatment also appeared to exacerbate the negative effects of incubation at 301C as judged by the high among-clutch variance of incubation length in the 28/30 treatment and relatively low survival of eggs from both the 28/30 and 30/30 treatments. Clutch effects Genetic and environmental components of clutch (maternal) effects have a wide spectrum of influence on development and hatchling phenotypes (Sinervo and Adolph, 94; Rhen and Lang, 98). Because selection acts on the phenotype, understanding the nature of direct clutch effects and the interactions between clutch and treatment and other environmental variables is important from an evolutionary perspective (West- Eberhard, 2003). This perspective can only be obtained from experimental designs that include clutch as a factor and in which clutches are replicated within treatments (e.g. Phillips and Packard, 94; Rhen and Lang, 98; Andrews et al., 2000; Andrews and Donoghue, 2004). Although small clutch size precludes such designs for many reptiles (e.g. Kearney and Shine, 2004), the large clutch sizes of chameleons facilitated testing for clutch effects. In this study, for example, when overall models were significant, clutch tended to explain more variation in dependent variables than treatment. Further insights into results of experimental manipulation of incubation temperature were gained by examination of clutch - treatment interactions. Such interactions for incubation length were the result of the differential sensitivity of individual clutches to high temperature per se and to treatments in which the incubation temperature was increased or decreased at the end of organogenesis. Clutch treatment interactions for body mass at 60 and 90 d were the result of two clutches in which the highest growth rates were exhibited in the 28/28 treatment and three clutches in which the highest growth rates were exhibited in the 28/30 treatment. Such a clutch-based variation in reaction norms will affect fitness apart from what appear to

11 INCUBATION TEMPERATURE AND CHAMELEON PHENOTYPE 445 be optimal phenotypes on the basis of mean treatment values. CONCLUSION Establishing a connection between environmentally induced phenotypes and fitness has proven to be a challenging undertaking. Most progress has been made with regard to temperature-dependent sex determination (Valenzuela and Lance, 2004; Warner and Shine, 2008). The reason is that sex is irreversibly determined and the two resultant phenotypes are discrete. In contrast, response to the incubation environment by most phenotypic traits is quantitative in nature and not necessarily permanent. Transitory effects make it extremely difficult to test the hypothesis of fitness benefit. Modulation of hatchling phenotypes by temperature, for example, may simply represent an immediate response of developmental processes to temperature that are selection neutral because of compensatory behavioral and physiological responses of individuals once they become active neonates (Radder et al., 2007). Results of studies reported here further highlight the complexities associated with the assessment of a phenotype fitness connection. ACKNOWLEDGMENT I thank the Morris Animal Foundation for funding, Jefferson Cox, Courtney Culp, Lydia Noble, Scott Parker, Harry Schwend, and Brian Siegel for technical assistance, Raju Radder for comments on the article, and Michael Leonard for advice on chameleon husbandry. The Virginia Tech Animal Care Committee approved the Research Protocol for this study in April LITERATURE CITED Andrews RM Embryonic development. In: Deeming DC, editor. Reptilian incubation: environment, evolution, and behaviour. Nottingham: Nottingham University Press. p Andrews RM Incubation temperature and sex ratio of the veiled chameleon (Chamaeleo calyptratus). J Herpetol 39: Andrews RM Effects of temperature on embryonic development of the veiled chameleon, Chamaeleo calyptratus. Comp Biochem Physiol 148A: Andrews RM, Donoghue S Effects of temperature and moisture on embryonic diapause of the veiled chameleon (Chamaeleo calyptratus). J Exp Zool 301A: Andrews RM, Mathies T, Qualls CP, Qualls FJ Rates of embryonic development of Sceloporus lizards: do cold climates favor rapid development? Copeia 1999: Andrews RM, Mathies T, Warner D Effect of incubation temperature on morphology, growth, and survival of juvenile Sceloporus undulatus. Herpetol Monogr 14: Andrews RM, Díaz-Paniagua C, Marco A, Portheault A Developmental arrest during embryonic development of the common chameleon (Chamaeleo chamaeleon) in Spain. Physiol Biochem Zool 81: Birchard GF, Reiber CL Effect of temperature on growth, metabolism, and chorioallantoic vascular density of developing snapping turtles (Chelydra serpentina). Physiol Zool 65: Booth DT Influence of incubation temperature on hatchling phenotype in reptiles. Physiol Biochem Zool 79: Braña F, Ji X Influence of incubation temperature on morphology, locomotor performance, and early growth of hatchling wall lizards (Podarcis muralis). J Exp Zool 268: Burger J Effects of incubation temperature on behavior of hatchling pine snakes: implications for reptilian distribution. Behav Ecol Sociobiol 28: Cuadrado M, Martín J, López P Camouflage and escape decisions in the common chameleon Chamaeleo chamaeleon. Biol J Linn Soc 72: Deeming DC Post-hatching phenotypic effects of incubation in reptiles. In: Deeming DC, editor. Reptilian incubation: environment, evolution, and behaviour. Nottingham: Nottingham University Press. p Díaz-Paniagua C Effect of cold temperature on the length of incubation of Chamaeleo chamaeleon. Amphibia- Reptilia 28:1 6. Downes SJ, Shine R Do incubation-induced changes in a lizard s phenotype influence its vulnerability to predators? Oecologia 120:9 18. Dufaure JP, Hubert J Table de développement du lézard vivipare: Lacerta (Zootoca) vivipara Jacquin. Arch Anat Microsc Morphol Exp 50: Elphick MJ, Shine R Longterm effects of incubation temperature on the morphology and locomotor performance of hatchling lizards (Bassiana duperreyi, Scincidae). Biol J Linn Soc 63: Flatt T, Shine R, Borges-Landaez PA, Downes SJ Phenotypic variation in an oviparous montane lizard (Bassiana duperreyi): the effects of thermal and hydric incubation environments. Biol J Linn Soc 74: Hare JR, Whitworth E, Cree A Correct orientation of a hand-held infrared thermometer is important for accurate measurement of body temperatures in small lizards and tuatara. Herpetol Rev 38: Janzen FJ, Paukstis GL Environmental sex determination in reptiles: ecology, evolution, and experimental design. Q Rev Biol 66: Kearney M, Shine R Developmental success, stability, and plasticity in closely related parthenogenetic and sexual lizards (Heteronotia, Gekkonidae). Evolution 58: Muth A Physiological ecology of desert iguana (Dipsosaurus dorsalis) eggs: temperature and water relations. Ecology 61: Necas P Chameleons: nature s hidden jewels. Frankfurt am Main: Edition Chimaira. Packard GC, Packard MJ The physiological ecology of reptilian eggs and embryos. In: Gans C, Huey RB, editors. Biology of the reptilia, ecology B, defense and life history, Vol. 16. New York: Alan R. Liss, Inc. p

12 446 ANDREWS Phillips JA, Packard GC Influence of temperature and moisture on eggs and embryos of the white-throated savannah monitor Varanus albigularis: implications for conservation. Biol Conservation 69: Qualls CP, Andrews RM Cold climates and the evolution of viviparity in reptiles: cold incubation temperatures produce poor quality offspring. Biol J Linn Soc 67: Qualls FJ, Shine R Post-hatching environment contributes greatly to phenotypic variation between two populations of the Australian garden skink, Lampropholis guichenoti. Biol J Linn Soc 71: Radder RS, Warner DA, Shine R Compensating for a bad start: catch-up growth in juvenile lizards (Amphibolurus muricatus, Agamidae). J Exp Zool 307A: Rhen T, Lang JW Among-family variation for environmental sex determination in reptiles. Evolution 52: Rhen T, Lang JW Phenotypic effects of incubation temperature in reptiles. In: Valenzuela N, Lance V, editors. Temperature-dependent sex determination in vertebrates. Washington, DC: Smithsonian Books. p SAS Institute, Inc SAS/STAT user s guide. Cary, NC: Statistical Analysis Systems Institute, Inc. Schmidt W Chamaeleo calyptratus: the Yemen chameleon. Münster: Matthias Schmidt Publications. Shine R, Elphick MJ 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: Shine R, Elphick MJ, Harlow PS The influence of natural incubation environments on the phenotypic traits of hatchling lizards. Ecology 78: Sinervo B, Adolph SC Growth plasticity and thermal opportunity in Sceloporus lizards. Ecology 75: Sokal RR, Rohlf FJ Biometry: the principles and practice of statistics in biological research. New York: W. H. Freeman and Co. Valenzuela N, Lance V, editors Temperature-dependent sex determination in vertebrates. Washington, DC: Smithsonian Books. Vanhooydonck BR, Van Damme TJ Evolutionary tradeoffs in locomotor capacities in lacertid lizards: are splendid sprinters clumsy climbers? J Evol Biol 14: Vanhooydonck BR, Van Damme TJ, Van Dooren M, Bauwens D Proximate causes of intraspecific variation in locomotor performance in the lizard Gallotia galloti. Physiol Biochem Zool 74: Warner DA, Andrews RM Laboratory and field experiments identify sources of variation in phenotypes and survival of hatchling lizards. Biol J Linn Soc 76: Warner DA, Shine R The adaptive significance of temperature-dependent sex determination in a reptile. Nature 451: West-Eberhard MJ Developmental plasticity and evolution. Oxford: Oxford University Press.