Natural History Note

vol. 176, no. 4 the american naturalist october 2010 Natural History Note The Physiological Basis of Geographic Variation in Rates of Embryonic Development within a Widespread Lizard Species Wei-Guo Du, 1,2,* Daniel A. Warner, 3 Tracy Langkilde, 4 Travis Robbins, 4,5 and Richard Shine 2 1. Hangzhou Key Laboratory for Animal Evolution and Adaptation, College of Biological and Environmental Sciences, Hangzhou Normal University, 310036 Hangzhou, People s Republic of China; 2. School of Biological Sciences, A08, University of Sydney, Sydney, New South Wales 2006, Australia; 3. Department of Ecology, Evolution and Organismal Biology, Iowa State University, Ames, Iowa 50011; 4. Department of Biology, Pennsylvania State University, University Park, Pennsylvania 16802; 5. Department of Integrative Biology, University of South Florida, Tampa, Florida 33620 Submitted March 28, 2010; Accepted June 16, 2010; Electronically published August 18, 2010 abstract: The duration of embryonic development (e.g., egg incubation period) is a critical life-history variable because it affects both the amount of time that an embryo is exposed to conditions within the nest and the seasonal timing of hatching. Variation in incubation periods among oviparous reptiles might result from variation in either the amount of embryogenesis completed before laying or the subsequent developmental rates of embryos. Selection on incubation duration could change either of those traits. We examined embryonic development of fence lizards (Sceloporus undulatus)from three populations (Indiana, Mississippi, and Florida) that occur at different latitudes and therefore experience different temperatures and season lengths. These data reveal countergradient variation: at identical temperatures in the laboratory, incubation periods were shorter for lizards from cooler areas. This variation was not related to stage at oviposition; eggs of all populations were laid at similar developmental stages. Instead, embryonic development proceeded more rapidly in cooler-climate populations, compensating for the delayed development caused by lower incubation temperatures in the field. The accelerated development appears to occur via an increase in heart mass (and, thus, stroke volume) in one population and an increase in heart rate in the other. Hence, superficially similar adaptations of embryonic developmental rate to local conditions may be generated by dissimilar proximate mechanisms. Keywords: countergradient variation, incubation period, heart rate, heart size, metabolic rate, Sceloporus undulatus. Introduction Developmental plasticity provides an important mechanistic explanation for geographic variation in phenotypes. In a wide range of organisms, the conditions under which an embryo develops alter the organism s phenotype in later * Corresponding author; e-mail: dwghz@126.com. Am. Nat. 2010. Vol. 176, pp. 522 528. 2010 by The University of Chicago. 0003-0147/2010/17604-52044$15.00. All rights reserved. DOI: 10.1086/656270 life (Deeming 2004; Watkins and Vraspir 2006). Given the sensitivity of developmental trajectories to local conditions, one of the most intriguing questions involves conservatism in phenotypic traits in wide-ranging species. Such conservatism often reflects countergradient variation, which occurs when genetic influences on a phenotype oppose environmental influences. For example, in both fruit flies (Drosophila serrata) and turtles (Chelydra serpentina), incubation periods at controlled temperatures are shorter for high-latitude populations than they are for low-latitude conspecifics (Ewert 1985; Liefting et al. 2009), canceling out the effects of environmentally driven thermal factors on the timing of hatching. The mechanistic basis of such shifts in developmental rates remains poorly understood. One possible mechanism may involve the thermal sensitivity of enzymatic activity. For example, in Atlantic salmon (Salmo salar), different forms of the trypsin isozyme can function at different temperature ranges and, in turn, regulate growth rate (Rungruangsak-Torrissen et al. 1998). Similar switches among alternative physiological pathways might enable embryonic developmental rates to adapt to local thermal conditions. Geographic variation in the duration of incubation of eggs in oviparous ectotherms offers an excellent model system with which to explore these issues. We expect incubation periods to be under strong selection because prolonged incubation (and development) increases the embryo s duration of exposure to nest conditions (e.g., vulnerability to predator attack or lethal extremes of temperature or moisture) and modifies the date of hatching (a critical determinant of hatchling fitness in many reptile species; Olsson and Shine 1997; Moreira and Barata 2005; Brown and Shine 2006; Warner and Shine 2007). What proximate mechanisms might adjust incubation periods? Plausible contenders include variation in (1) the degree of embryogenesis completed before ovi-

Developmental Rate of Embryonic Lizards 523 position (developing eggs retained in utero for a longer duration would require less subsequent time in the nest; Warner and Andrews 2003; Radder et al. 2008), (2) degree of offspring development before hatching (a less developed offspring might be able to hatch earlier; Shine and Olsson 2003; Colbert et al. 2010), (3) maternal selection of nest sites (hotter nests will accelerate embryonic development; Shine 2004; Angilletta et al. 2009), and (4) rates of embryonic development at any given temperature (fasterdeveloping embryos will complete development sooner). Cardiac output is an important determinant of the rate of embryogenesis because it plays a critical role in nutrient and oxygen delivery during development (Birchard and Reiber 1996; Birchard and Deeming 2004; Tazawa 2005). Variables such as heart size, heart rate, and stroke volume are related to cardiac output as well as to rates of metabolic expenditure and embryonic development (Faber et al. 1974; Kam 1993; Burggren and Keller 1997; Pearson et al. 2000). Indeed, in reptiles, the completion of development requires a fixed number of heart beats by embryos (Du et al. 2009). Thus, specific mechanisms to accelerate embryogenesis might include an acceleration of heart rate relative to temperature or an increase in heart size (and thus cardiac output; Kam 1993; Du et al. 2009). Complicating the picture still further, a capacity for thermal acclimation by embryos (Huey and Berrigan 1996; Liefting et al. 2009) might shift heart rates relative to incubation conditions as a direct environmental effect. Importantly, the proximate mechanisms listed above are not mutually exclusive: incubation periods may be adjusted via interactions among multiple factors, or different mechanisms may occur in different parts of the geographic range of a species. We tested the relationship between cardiac output and developmental rates across a latitudinal gradient, using the eastern fence lizard (Sceloporus undulatus), a mediumsized (snout-vent length up to 80 mm) species found along forest-edge habitats in North America (Tinkle and Ballinger 1972). Its phylogeographic history is complex, with multiple evolutionary shifts toward distinctive ecomorphs in various parts of the range (Leache and Reeder 2002; Leache 2009). Under identical incubation temperature regimes, the incubation period is shorter for lizards from northern populations than it is for those from southern populations (Oufiero and Angilletta 2006). This geographic divergence cannot be attributed to the amongpopulation differences in offspring size, because experimental manipulation of egg sizes does not alter incubation periods (Oufiero and Angilletta 2006). To clarify the mechanism(s) responsible for this example of countergradient variation in incubation period, we compared embryonic development and cardiac output among three widely separated populations of S. undulatus. If geographic divergence in incubation periods is driven by variation in the degree of uterine retention of developing eggs, we expect higher-latitude females to produce eggs with embryos at later stages. If developmental rates differ, we expect divergence in traits such as heart rate relative to temperature, heart mass of hatchlings relative to body mass, and/or the extent of thermal acclimation in developmental rates. Material and Methods Animal Collection and Husbandry We captured gravid female Scleoporus undulatus from three populations that span almost the entire latitudinal range for this species, in the eastern portion of its range. The northern, central, and southern populations were collected from Monroe County, Indiana (39 17 N, 86 50 W); Teasdale County, Mississippi (34 16 N, 90 02 W); and Hillsborough County, peninsular Florida (27 45 N, 82 15 W), respectively. All captured lizards were transferred to the laboratory at Iowa State University, where they were housed in glass terraria (650 mm # 300 mm # 300 mm) filled with 10 mm of moist sand and kept in a room with a temperature of 22 1 C and a light cycle of 12L : 12D (on at 0700 hours, off at 1900 hours). A 100-W light bulb suspended 50 mm above the terrarium provided supplementary heating from 0800 to 1600 hours. Food (crickets dusted with vitamins and minerals) and water were provided ad lib. Egg Collection and Incubation Each terrarium housed three gravid females and was checked at least three times a day for freshly laid eggs. Our regular checks for eggs and palpation of females ensured that we were able to assign maternity to clutches. Once found, the eggs were weighed ( 0.001 g) immediately, and one egg from each clutch was randomly selected to identify the embryonic stage at oviposition on the basis of Sanger et al. s (2008) classification scheme. The remaining eggs were individually incubated in 64-mL glass jars filled with moist vermiculite ( 150 kpa) and covered with plastic wrap (sealed with a rubber band). The jars were then assigned evenly to two thermal regimes, constant 25 C and constant 28 C, in a split-clutch design. A total of 15, 16, and 10 clutches of eggs from different females were collected for the northern, central, and southern populations, respectively. Heart Rate, Heart Mass, and Hatchling Size We measured heart rates of embryos approximately halfway through the total incubation period (day 25 for 28 C

524 The American Naturalist and day 35 for 25 C). The number of clutches used in the heart rate experiment was 12, 11, and 8 for the northern, central, and southern populations, respectively. Following a 2-h period in the relevant incubator to attain thermostability, heart rates (beats per minute [bpm]) were measured at four test temperatures (20, 25, 30, and 33.5 C) using an infrared heart rate monitor (Buddy System, Avian Biotech; see detailed procedures in Du et al. 2009). These four temperatures spanned the major range of temperatures experienced by eggs in field nests (Angilletta et al. 2009). On the day of hatching, snout-vent length (SVL) and body mass of hatchlings were determined using a ruler ( 1 mm) and a balance ( 0.01 g), and some hatchlings from each population (eight, seven, and seven clutches of individuals from the northern, central, and southern populations, respectively) were euthanized and their hearts were dissected out. Fresh heart mass was weighed using a 0.0001-g balance. Heart masses during earlier embryo stages were not measured because the hearts were too small for accurate measurements. Therefore, our analyses of heart mass in newly hatched lizards assume that heart size of hatchlings reflects the relative heart size during earlier stages of development (i.e., the times when heart rate was quantified). ( F2, 76 p 10.0, P!.001). At each incubation temperature, eggs from the lower-latitude population took longer to hatch than did those from higher-latitude areas (southern 1 central 1 northern; fig. 1). Developmental stage of embryos at oviposition did not vary significantly among populations (mean SE p 3.4 0.2 for all three pop- 2 ulations; x p 1.25, P p.53, n p 10 16 eggs [one per clutch] per population). Heart Rates, Heart Mass, and Hatchling Size Heart rates strongly depended on test temperatures ( F p 4,652.9, P!.00001), differed among the three 3, 168 Statistical Analysis We used the software package STATISTICS 6.0 to analyze data. Normality of distributions and homogeneity of variances were tested using the Kolmogorov-Smirnov test and Bartlett s test, respectively. To avoid pseudoreplication, all analyses were based on clutch means. Two-way ANOVAs with population and incubation treatment as factors were conducted to explore differences in incubation period between populations as well as thermal regimes. An ANCOVA with initial egg mass or hatchling body mass as a covariate was used to examine among-population variation in hatchling size or heart mass. Repeated-measures ANOVAs were used to test for among-population differences and thermal effects on heart rates. Tukey s post hoc multiple comparisons were used to detect differences among populations. Because the data were not normally distributed, a median test was used to compare embryonic stages at oviposition among populations. Results Incubation Period Incubation period was affected by temperature (F1, 76 p 2,760.9, P!.00001), population of origin ( F2, 76 p 107.4, P!.00001), and the interaction between these two factors Figure 1: Incubation periods of eggs at two constant temperatures (25 and 28 C) in Sceloporus undulatus from northern, central, and southern populations (Indiana, Mississippi, and Florida, respectively). Incubation periods differed among the populations and decreased with increasing latitude at both incubation regimes. Data are expressed as mean SE. All means were statistically different (Tukey s post hoc test). Numbers above error bars represent the number of clutches used for each population.

Developmental Rate of Embryonic Lizards 525 populations ( F2, 56 p 5.06, P p.01), and depended on incubation temperatures ( F1, 56 p 37.08, P!.00001), with no significant interaction between population and incubation temperature ( F2, 56 p 0.75, P p.48; fig. 2). At any given test temperature, the heart rates of embryos incubated at 25 C were higher than those of embryos incubated at 28 C (in all populations). The heart rates of embryos from the central population were higher than those of embryos from the northern and southern populations (fig. 2). The mean temperature coefficient (Q 10 ) values of heart rates for all populations were 3.0, 2.0, and 2.2 at temperature ranges of 20 25 C, 25 30 C, and 30 33.5 C, respectively. Fresh heart mass of hatchlings was not affected by incubation temperature ( F1, 15 p 0.87, P p.37) or the interaction between temperature and population (F2, 15 p 0.88, P p.43). However, hatchling heart mass differed among populations, even after the effect of hatchling size was statistically removed ( F2, 15 p 10.77, P p.001). Relative to body mass, hearts of lizards from the northern population were heavier than those of individuals from the central and southern populations (adjusted means SE: northern, 2.7 0.1 mg, n p 8; central, 1.9 0.1 mg, n p 7; southern, 1.9 0.1 mg, n p 7). Mean hatchling SVL and body mass differed significantly among populations, with greater hatchling size in the northern and central populations than in the southern population (Wilks F4,148 p 9.10, P!.00001); neither SVL nor mass of hatchlings was affected by incubation temperature (Wilks F2, 74 p 1.52, P p.22) or the interaction between temperature and population (Wilks F4,148 p 0.14, P p.97). After the data on hatchling size from the two incubation treatments were pooled, hatchling SVL averaged 25.19 0.15 mm ( n p 30), 25.18 0.14 mm ( n p 32), and 23.93 0.18 mm ( n p 20), and body mass averaged 0.53 0.01 g ( n p 30), 0.51 0.01 g ( n p 32), and 0.48 0.01 g ( n p 20), in the northern, central, and southern populations, respectively. Discussion As reported by previous studies on Sceloporus undulatus (Oufiero and Angilletta 2006; Niewiarowski and Angilletta 2008), we found significantly shorter incubation periods in lizards from higher-latitude populations than in those from lower-latitude populations, even when all of the eggs were incubated at the same temperatures. Given the strong negative direct effect of temperature on incubation period and decreasing ambient temperatures from the southern to the northern sites, the decreasing incubation periods of higher-latitude lizards appear to be an example of countergradient variation. Our data falsify the hypothesis that variation in incubation periods among populations was due to differences in developmental stage at laying, because developmental stages of embryos at oviposition did not differ significantly among populations. Our data also falsify the hypothesis Figure 2: Heart rates of lizard embryos (Sceloporus undulatus) from northern, central, and southern populations (Indiana, Mississippi, and Florida, respectively) incubated at 25 and 28 C. Heart rates were measured at 20, 25, 30, or 33.5 C approximately halfway through the incubation period. Data are expressed as mean SE.

526 The American Naturalist that geographic variation in incubation period stems from geographic variation in the degree of offspring development before hatching. That hypothesis predicts less developed offspring and thus smaller hatchlings in northern populations (due to earlier hatching) than in southern populations (Shine and Olsson 2003), but our observed trend is opposite to this predicted geographic cline. Also, both hatchling size and locomotor performance show relatively little geographic variation within this species (Niewiarowski and Angilletta 2008). Fence lizard embryos exhibit thermal acclimation to incubation temperature in heart rates, with low-temperature-incubated embryos showing higher heart rates than their high-temperature-incubated siblings (fig. 2). This partial compensation of metabolic rate increases developmental rates of embryos at low temperatures, allowing an embryo to hatch relatively early in the season even if it develops inside an unusually cool nest. Thermal acclimation of metabolic compensation has also been reported in the turtle Chelydra serpentina and the crocodile Crocodylus johnstoni (Whitehead 1987; Birchard and Reiber 1995), as well as in another population of S. undulatus (Angilletta et al. 2000). Previous work has suggested that thermal acclimation ability may differ among populations and be restricted to temperate rather than tropical animals (Tsuji 1988; Liefting et al. 2009). That prediction is based on the idea that compensatory evolution of developmental rate is more important for fitness in highly variable conditions and short growing seasons (Conover and Schultz 1995) than it is in more stable tropical areas that rarely experience cold nest temperatures (Liefting et al. 2009). Our data do not support this hypothesis. In S. undulatus, both temperate and subtropical populations showed thermal acclimation of cardiac function. It is possible that thermal regimes inside natural nests do not differ as greatly among populations as would be suggested by a comparison of air temperatures at these sites; nesting females may buffer the effects of geographically variable climatic conditions such that embryos experience similar thermal environments inside nests across a wide geographic range (Morjan 2003; Doody et al. 2006; Angilletta et al. 2009). In future studies, fieldwork to measure temperature regimes inside nests across a latitudinal gradient would be of great interest. Given the similarity among populations in acclimation ability and stages of embryonic development at laying, why did eggs from the northern and central populations hatch sooner than eggs from the southern population? The answer lies in developmental rates of embryos, but the plausible mechanism allowing that acceleration of development differs between the two short-incubation-period populations. Embryos from the northern population had relatively large hearts, thereby increasing cardiac output (stroke volume; Saari et al. 1999) and, therefore (at least potentially), metabolic and developmental rates. In contrast, embryos from the central population had hearts that were similar in size to those of the southern lizards, and they may have increased their developmental rates by increasing their heart rates (fig. 2). These conclusions are supported by the fact that, in reptiles, the completion of development requires a fixed number of heartbeats by embryos (Du et al. 2009). We do not know why these two populations have solved the same problem in two different ways (perhaps increased heart rate alone is insufficient to accelerate development enough in cool conditions such as those experienced in more northern populations; Parker and Andrews 2007), but our results suggest that disparate proximate mechanisms may underlie superficially similar phenotypic traits. More generally, embryonic adaptations are an underutilized and underappreciated resource for evolutionary biologists. The embryonic stage provides exciting opportunities to identify the proximate mechanisms underlying interspecific and intraspecific variation in life-history traits. Much of the variance in life-history traits that are expressed in later stages of the organism s life (such as age at maturation and fecundity) reflects environmentally induced plasticity, particularly in response to food availability (Bonnet et al. 2001; Du 2006; Illera and Diaz 2006). However, developmental timing is likely under strong and geographically divergent selection pressures in many species (Sternberg and Grinkov 2006; Warner and Shine 2007; Syrjanen et al. 2008), facilitating comparative analysis of the adaptive mechanisms by which local selective forces act on embryogenesis overall and on developmental rates in particular. Our study identifies variation in embryonic heart rate and size as mechanisms that can generate geographic variation in rates of growth and development. Acknowledgments We thank M. Angilletta, A. Bronikowski, C. Chandler, N. Freidenfelds, F. Janzen, T. S. Schwartz, J. Ward, and H. Ye for their assistance in the field or the laboratory. We thank P. H. Niewiarowski and two anonymous reviewers for their constructive comments on the manuscript. W.-G.D. thanks F. Janzen for hosting his stay in Iowa and making this work possible. The study was approved by the Institutional Animal Care and Use Committee at Iowa State University. Animals were collected under appropriate state permits (Indiana: 09-0103, Mississippi: 0511091, and Florida: WX05107). We thank M. Angilletta, R. Kiihnl, and H. Mushinsky for facilitating our collection of animals in Indiana, Mississippi, and Florida, respectively. This work was supported by grants from the Natural Science Foundation of China, the University of Sydney (to W.-G.D.), and the

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