Canadian Journal of Zoology. Thermal consequences of subterranean nesting behavior in a prairie-dwelling turtle

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1 Canadian Journal of Zoology Thermal consequences of subterranean nesting behavior in a prairie-dwelling turtle Journal: Canadian Journal of Zoology Manuscript ID cjz r1 Manuscript Type: Article Date Submitted by the Author: 18-Oct-2016 Complete List of Authors: Tucker, Charles; Missouri State University, Biology Strickland, Jeramie; Upper Mississippi River National Wildlife and Fish Refuge Delaney, David; U.S. Army Construction Engineering Research Laboratory Ligon, Day; Missouri State University, Biology Keyword: Terrapene ornata, ornate box turtle, phenotypic plasticity, incubation, THERMOREGULATION < Discipline, nest site selection

2 Page 1 of 35 Canadian Journal of Zoology 1 Title: Thermal consequences of subterranean nesting behavior in a prairie-dwelling turtle Charles R. Tucker, Jeramie T. Strickland, David K. Delaney, and Day B. Ligon CRT* (Corresponding Author) and DBL: Department of Biology, Missouri State University, 901 South National Avenue, Springfield, Missouri JTS: Upper Mississippi River National Wildlife and Fish Refuge, 7071 Riverview Road, Thomson, Illinois DKD: U.S. Army Construction Engineering Research Laboratory, P.O. Box 9005, Champaign, Illinois *Present Address: Red Lake Wildlife Management Area, P.O. Box 100, Roosevelt, Minnesota 56673; crtucker2@gmail.com; telephone: 1(218) ; FAX: 1(218) Keywords: Terrapene ornata ornate box turtle phenotypic plasticity incubation thermoregulation nest site selection

3 Canadian Journal of Zoology Page 2 of 35 2 Thermal consequences of subterranean nesting behavior in a prairie-dwelling turtle C.R. Tucker, J.T. Strickland, D.K. Delaney, and D.B. Ligon Abstract: Many oviparous reptiles deposit eggs in excavated nest chambers, and the location and depth at which eggs are laid can affect predation risk, incubation duration, mortality rates, and hatchling phenotype. Among turtles, nest depth also influences incubation conditions of some large-bodied species, but nest depth is generally expected to vary less among small-bodied species. We monitored nesting behavior of ornate box turtles (Terrapene ornata (Agassiz, 1857)) for two seasons in Illinois. We used direct observations to confirm that, among 31 nesting events, 6 females oviposited while beneath the substrate surface. Furthermore, comparisons of body length to nest depth indicated that 5 additional females likely also constructed nests while buried. Nests laid while females were underground were deeper on average than other nests (16.7 versus 11.2 cm), and while average nest temperatures were similar between groups, temperature fluctuations and maximum temperatures were lower among nests that were laid while females were underground. Subterranean oviposition appears to have moderated incubation temperatures by allowing females to deposit eggs at greater depths than would be possible from the surface. This little-documented behavior may be a mechanism for this species to influence the incubation environment, which in turn may influence hatchling phenotypes.

4 Page 3 of 35 Canadian Journal of Zoology 3 Introduction Nest site selection has important and varied fitness implications for oviparous animals (Bullmer and Bull 1982; Weisrock and Janzen 1999; Refsnider and Janzen 2012). Among species that leave clutches to develop in subterranean chambers, the nesting process includes a suite of energetically costly activities, including searching for a suitable nest site, excavating a cavity, and then covering and camouflaging the nest. Additionally, females are usually exposed to elevated predation risks during nesting forays (Spencer and Thompson 2003), and location can also affect predation risk to offspring during both incubation and hatchling dispersal (Wood and Bjorndal 2000; Spencer and Thompson 2003). Finally, the biotic and abiotic characteristics of a nest site may influence incubation duration and hatchling phenotype (Ewert et al. 2005; Freedberg et al. 2008). Oviparous reptiles have flexibility in selecting the geographical location of their nests, and this decision can determine the conditions to which eggs are exposed (Doody et al. 2006). Females also have the capacity to select the depth at which they deposit a clutch, which can influence soil moisture, mean temperature, the magnitude of temperature fluctuations (Packard et al. 1987; Doody et al. 2006), and risk of nest predation (Leighton et al. 2009; Doody et al. 2015). Most species of turtle follow a fairly consistent nesting pattern, wherein a female selects a nest site, positions her plastron on the surface, uses her hind limbs to excavate a cavity, deposits eggs, fills the cavity, and leaves the site (Miller et al. 2003). Adherence to this pattern may limit the depths to which females are able to excavate (Ehrenfeld 1979), and consequently affect the range of incubation conditions available to females depositing eggs from the surface, especially for small-bodied species (Wilson 1998; Refsnider et al. 2013). Some species may also create body pits (Hailman and Elowson 1992) or become partially buried during nesting (Ehrenfeld 1979),

5 Canadian Journal of Zoology Page 4 of 35 4 but there are few reports of complete burial during the nesting process (Christensen et al. 1985; Iverson 1990; Lee 2012). The ornate box turtle (Terrapene ornata (Agassiz, 1857)) is a small emydid turtle that has a large geographic range but typically occurs in prairie habitats dominated by sandy soils (Converse et al. 2002; Dodd 2002). We monitored an ornate box turtle population for two nesting seasons and observed several instances of females ovipositing while completely below the substrate surface, a behavior that allowed these females to oviposit deeper underground than would otherwise be possible. We hypothesized that nests would be deeper and average nest temperatures and daily temperature fluctuations would be lower in nests that were excavated by subterranean females. In addition to testing this prediction, we also include information on clutch size and hatching rates, as well as observations on the nesting habits of this prairie-dwelling species. Materials and methods Study Site We conducted our study on two remnant sand prairies at the Upper Mississippi River National Wildlife and Fish Refuge in northwestern Illinois. Thomson-Fulton Sand Prairie ( N, W) is 146 ha and is bordered to the west by a slough of the Mississippi River and to the east by a dirt road and railroad tracks. The site is dominated by native sand prairie vegetation and has been identified as a high-quality vegetation community (Ebinger et al. 2006). There is also a corridor of remnant prairie associated with a railroad rightof-way that runs parallel to the site. A 16-ha pine plantation separates this prairie from another remnant prairie to the south, and the two prairies are connected by a narrow corridor of remnant

6 Page 5 of 35 Canadian Journal of Zoology 5 prairie associated with a bicycle trail and the railroad tracks. Study animals have been observed using the pine plantation and both prairies as well as the remnant corridor between them. The second study site is a 6.9-ha enclosure at the Lost Mound Unit of the Upper Mississippi River National Wildlife and Fish Refuge ( N, W) that was constructed in 2010 as a soft release site for an ornate box turtle reintroduction to the Unit. The site was an active military installation from until management was transferred to the United States Fish and Wildlife Service. Military use prevented conversion of the site to row crops (Ebinger et al. 2006), and as a result the Lost Mound Unit is the largest contiguous remnant sand prairie in Illinois. Ornate box turtles were historically common at the site, but decades of military activity nearly extirpated them (McCallum and Moll 1994). Study Animals In spring 2011 and 2012, we hand-captured (or re-captured for transmitter replacement) 34 reproductively mature female ornate box turtles (mass = 348 ± 48 g, mean ± sd) and mounted radio transmitters ( MHz, Advanced Telemetry Systems, Isanti, Minnesota) to costal scutes 2 and 3 and an occasional adjacent scute using 5-minute epoxy (Bernstein and Black 2005). Transmitters and epoxy were a combined 4 6% of turtle mass. The study population included 25 resident females at Thomson Sand Prairie, and 1 resident and 8 translocated females at the Lost Mound Unit. Due to transmitter failure and removal by predators, only 18 turtles at Thomson Sand Prairie and 6 at Lost Mound were monitored in both years of the study. Locating and Documenting Nests During May and June in 2011 and 2012, turtle activity was monitored to detect nesting

7 Canadian Journal of Zoology Page 6 of 35 6 activity using an automated radio telemetry system (Tucker et al. 2014). The telemetry system consisted of 5 automated receiving towers that recorded signal strengths from each turtle s transmitter at 2-minute intervals. Because any movement, including slight changes in orientation, can cause large changes in the magnitude of signals reaching stationary receiving stations, temporal changes in signal strength were used as indices of turtle activity (Cochran and Lord 1963; Tucker et al. 2014). Because ornate box turtles are diurnal unless engaged in nesting activity (Legler 1960; Doroff and Keith 1990), night activity by females during the nesting season was used as an indicator of likely nesting behavior. Data from the automated telemetry system were viewed nightly in near real-time during the nesting season, and when the presence of nocturnal activity indicated potential nesting behavior, females were located using a hand-held radio receiver (R2179, Advanced Telemetry Systems, Isanti, MN). When nesting females were located, their positions were recorded using a GPS and marked with survey flags, and visual determinations of the subterranean status of females were made. These sites were then revisited the following morning and cavities containing eggs were carefully excavated. As eggs were removed from the cavity, the depth of the top and bottom of the clutch were recorded. Eggs were returned to the cavity at the same depth and orientation from which they were removed, and temperature data loggers (Thermochron ibutton model DS1922L, Maxim Integrated Products, Inc., Sunnyvale, CA) were placed directly above and below each clutch so that data loggers were in contact with the uppermost and bottommost eggs, respectively. Data loggers were coated in rubber (Performix Plastidip International, Blaine, MN) and were set to record temperatures hourly. Nest protectors (1.25-cm wire mesh, covering the top of the site and extending to a minimum depth of 15 cm on all sides) were also placed over nests to reduce predation.

8 Page 7 of 35 Canadian Journal of Zoology 7 This project coincided with conservation efforts intended to increase the presence of this species in northwestern Illinois. To this end, nest sites were monitored throughout incubation to document predation attempts. Such attempts generally included obviously displaced sand, scratch marks, and/or tunnels under the wire mesh. Because of the small size of this population (Kuo and Janzen 2004) and concerns that exceptionally warm and dry conditions of 2012 would reduce hatching success by desiccating eggs, approximately 4 L of water was applied to each nest four times throughout the incubation period. Nests were excavated on 1 October 2011 and 18 September 2012, and data loggers were recovered from the nest cavities. Egg shell fragments and unhatched eggs were collected to assess hatching success rates. Cavities were excavated to depths of 80 cm, and the depths at which hatchlings were encountered were recorded. Hatchlings were then reburied in the same orientation and at the same depth at which they were found. Often, only a single focal observation was used to make determinations of a female s subterranean status due to time constraints during nights when nesting occurred. Because females may have been observed before or after underground portions of nest construction occurred, some underground nesting behavior may not have been documented at the time that nests were located. To address this limitation, in 2012 infrared time-lapse cameras (PC 900, Reconyx, Holmen, WI) were placed at nest sites and set to record photographs at 1-minute intervals. Due to the generally warm and dry conditions of 2012, many females nested simultaneously during a single rain event in early June. As a result, personnel and equipment resources were insufficient to adequately monitor all nesting females, and the construction of several nests was not photographed. Additionally, it is possible that among monitored nests, we did not deploy cameras early enough in the nesting process to document every instance of subterranean behavior.

9 Canadian Journal of Zoology Page 8 of 35 8 To corroborate visual and photographic evidence, or in lieu of such data when none were available, the maximum depth of nests was compared to female body length. If the distance from the ground surface to the bottom of a nest cavity was greater than the female s body length, we inferred that a female was below the ground surface when oviposition occurred. Because cavities are constructed using hind limbs and heads are rarely extended during nesting, anterior appendages rarely contribute to the functional body length that would allow a female to remain visible from the surface while constructing a deep cavity. As a result, body length was calculated as the distance from the anterior edge of the carapace to the distal end of outstretched hind limbs. Because females do not orient vertically while constructing nest cavities, this body length measurement likely represents an over estimate of the maximum depths to which a female could excavate without going beneath the surface. Thus, three categories were identified: females that nested from the surface, females that were observed nesting underground, and females that were inferred to have nested underground based upon morphometric and nest depth measurements. In the single instance when retrospective determinations of oviposition method and visual observations did not agree, nesting behavior was scored based on the visual observations. When we failed to observe subterranean activity and body length measurements exceeded the maximum depth of a cavity, we assumed that females oviposited without burrowing. Conversely, females that we observed underground on the night of oviposition were classified as nesting underground without comparing nest depths and female body length. Females who were not observed completely below the surface, or who did not oviposit more deeply than body length measurements were classified as having nested from the surface even if they were observed to be partially (but not completely) buried during the nesting process.

10 Page 9 of 35 Canadian Journal of Zoology 9 Calculating Hatching Times Incubation duration varies inversely with incubation temperature, but the relationship is not well documented for ornate box turtles within the temperature ranges experienced by natural nests at this site (Legler 1960). Mean incubation temperatures have been shown to be reliable predictors of hatching times in other species (Kaska et al. 1998), and we calculated hatching times for turtles in this study using a binomial regression based on constant-temperature incubation trials conducted in 2003 (D. Ligon, unpublished data). Hatching times were estimated with the equation: =.. +. where D is incubation duration measured in days and c is the mean incubation temperature, expressed in degrees Celsius. Statistics Average incubation temperatures and depths at mid-point of the nest cavity were used to compare clutch depths and temperatures between females that nested from the surface and females that nested underground using Student s t-tests. Sustained temperatures as low as 32 C can reduce hatching rates in turtles (Du et al. 2007), but we observed short periods when temperatures were much higher than this. To determine if transient exposure to high temperatures reduces hatching rates, we used t-tests to compare cavity mid-depths between nests that had maximum incubation temperatures at the top of the clutch above and below 40 C, as even brief periods above this threshold are likely to cause embryonic death. Two-sample t-tests were also used to compare maximum incubation temperatures at the top of the clutch between nests created from the surface and those created while females were burrowed. We used one-way ANOVA

11 Canadian Journal of Zoology Page 10 of tests conducted on standard deviations of incubation temperatures to compare the magnitude of temperature fluctuations in nests constructed from the surface versus from a subterranean position. Least-squares means regressions were used to test relationships between: i) maximum incubation temperature and the number of unhatched eggs per clutch; ii) cavity mid-depth and mean incubation temperature; and iii) cavity mid-depth and maximum incubation temperature. All conclusions were based on a Type I Error Rate of Results Nesting Behavior In 2011 and 2012, we located 31 nests by remote telemetry monitoring. Of these, we visually confirmed six instances of subterranean oviposition, either by direct observation or from images collected by time-lapse cameras. Based on comparisons of nest depth and body length, we inferred that at least five additional females oviposited below the surface. Other females oviposited from the surface or while partially buried, and comparisons of nest depth and female body length generally corroborated our visual observations. Comparisons of nest depth and body length indicated that in all but a single nest, females that were observed underground could not have deposited eggs at such depths without burrowing below the surface. A single female engaged in at least 2.25 h of underground activity on the night that she nested, but a comparison of her body length to the depth of her nest cavity indicated she could have attained the same oviposition depth without subterranean oviposition, so it is unclear what her body position relative to the substrate surface was during oviposition. Nonetheless, this female was included in the confirmed subterranean category because both direct and photographic observations confirmed that she was below the surface. Compared to nests of females that oviposited

12 Page 11 of 35 Canadian Journal of Zoology 11 terrestrially, nests were deeper when females were visually verified to oviposit while buried (t 5 = 2.54, P = 0.044) and when females were inferred to have oviposited while buried based upon comparisons of body length and nest depth (t 4 = 3.56, P = 0.016). The nests of females observed and inferred to oviposit underground combined were also deeper than nests from females that oviposited from the surface (t 10 = 4.10, P = 0.001; Fig. 1). There were no significant differences in depth (t 4 = 0.63, P = 0.544) or temperature (t 2 = 0.53 P = 0.648) between nests of females confirmed and inferred to have oviposited underground. Nest Temperatures We placed data loggers in 14 nests in 2011 (2 data loggers subsequently failed) and in nine nests in The mean ± sd nest temperature among all nests was 27.9 ± 1.0 C in 2011 and 29.2 ± 0.5 C in An additional nine nests were located in 2012, but the eggs were removed from the field and incubated artificially as part of a head-start reintroduction program. Therefore, only nest depth and nesting behavior data were available for these nests (Fig. 1). Mean incubation temperatures were similar among all groups (Fig. 2), but maximum temperatures at the top of nest cavities were lower among nests deposited while females were underground (t 8 = 3.00, P = 0.008). Compared to nests of females that oviposited from the surface, nest temperature profiles exhibited less thermal variation when females were visually confirmed to oviposit underground (ANOVA; F 1,16 = 10.16, P = 0.006) and when females were inferred to have oviposited while buried (ANOVA; F 1,15 = 4.67, P = 0.048) (Figs. 3, 4). The nests of visually observed and inferred subterranean nesters combined also exhibited less variation in temperature than nests that were constructed from the surface (ANOVA; F 1,20 = 14.36, P = 0.001). There was no difference in thermal variation between the nests of females that were

13 Canadian Journal of Zoology Page 12 of visually observed versus inferred to have nested underground (ANOVA; F 1,7 = 0.42, P = 0.542). Mean temperature and precipitation norms for the incubation period (May August) from 1971 to 2000 were 19.8 C and 40.7 cm, respectively. Mean values for the same period in 2011 and 2012 were 21.5 C and 24.2 cm, and 22.2 C and 30.8 cm respectively (NOAA National Climactic Data Center, Fulton, IL, Lock and Dam 13, accessed 12 March 2013). During particularly warm portions of the incubation period, most nests experienced exceptionally high temperatures. Twenty of 23 nests recorded maximum temperatures at the top of the egg chamber of at least 35.0 C at some point during the incubation period, and seven clutches experienced maximum temperatures that exceeded 40.0 C (maximum = 43.3 C). Nests that had maximum temperatures above 40.0 C were shallower than other nests (t 6 = -4.74, P < 0.001) and none of the females who laid such nests exhibited subterranean nesting behavior. The number of unhatched eggs in a clutch did not correlate with maximum incubation temperature (R 2 = 0.90, slope = -0.07, P = 0.288). Among all nests, cavity mid-depth was not related to mean incubation temperature (R 2 = 0.90, slope = -0.07, P = 0.87), but was related to maximum temperature at the top of the clutch (R 2 = 0.43, slope = -0.48, P < 0.001) (Fig. 5). Nest Outcomes Hatching rates were generally high, with a cumulative hatching rate among nests incubated naturally over the two year study period of 83% (80% in 2011 and 87% in 2012). When nests from both study sites were excavated 1 October 2011, 10 live hatchlings were found distributed among four nests at a mean depth of 24 cm. Some hatchlings remained in the nest cavity (11 cm), while others were found much deeper (maximum = 38 cm), indicating that they had burrowed below the nest cavity after hatching. Also in 2011, 10 of 49 eggs from seven

14 Page 13 of 35 Canadian Journal of Zoology 13 different nests did not hatch. In one nest, all 4 eggs failed to hatch, while single eggs from 6 other clutches did not hatch. Eggshell fragments indicating fully hatched clutches were found in the remaining 7 nests in When naturally incubated nests from the 2012 season were excavated 18 September 2012, no hatchlings were found despite excavating a large area around each cavity to 80 cm depth. Also in 2012, only 2 of 39 eggs located upon nest excavation had failed to hatch. Discussion Nesting Behavior Based on observations of females that emerged from the substrate and then immediately began to fill cavities that were later found to contain eggs, as well as other females that oviposited deeply (up to 48% deeper than body length measurements) relative to their small body size, it is clear that females from this population sometimes oviposit while completely buried below the soil surface. However, other females oviposited from the surface or while only partially buried, indicating a great deal of variation in nest cavity construction occurs among female ornate box turtles. Typically, females who oviposited while underground were initially located under a thin layer of disturbed soil. Subterranean activity was visually evident from movements of the soil surface caused by the females, or by movements of small portions of the 22-cm transmitter antennas that were sometimes visible from the surface. Often, females burrowed beneath the surface and spent several hours underground excavating a cavity and ovipositing before returning to the surface to fill the cavity and manicure the site. We documented one female that exhibited underground behavior but deposited eggs at depths that could have been achieved from the

15 Canadian Journal of Zoology Page 14 of surface. Because behavioral observations were not continuous, it is possible that other females exhibited subterranean behavior but subsequently created shallow nests. Yellow mud turtles (Kinosternon flavescens (Agassiz, 1857)) are reported to sometimes burrow below the surface to deposit eggs and then remain underground with the clutch for several weeks during estivation (Christiansen et al. 1985, Iverson 1990). However, the adaptiveness or relatedness of these two behaviors (subterranean oviposition and remaining underground with the clutch) is not clear. Yellow mud turtles, like ornate box turtles, are relatively small turtles that nest in arid uplands and may benefit from the additional clutch depth afforded by subterranean oviposition. Furthermore, remaining underground with the clutch after oviposition may reduce predation and/or reduce egg desiccation rates (Iverson 1990). However, it is unclear whether subterranean nesting and subsequent nest tending evolved in this species as a mechanism for increasing nest success or is simply a consequence of the species proclivity for summer estivation. Because subterranean nesting is not coupled with estivation in ornate box turtles, its occurrence in this species seems likely a result of selection on nesting behavior. Nest Temperatures Mean incubation temperatures were similar across terrestrial and subterranean oviposition methods, but daily temperature fluctuations were reduced among clutches deposited while the female was underground. Therefore, reduced daily temperature extremes may be the most significant thermal consequence of subterranean oviposition. Even transient exposure to high incubation temperatures can be sufficient to kill embryos (Miller and Ligon 2014), so selection is likely strong for behaviors that reduce temperature extremes. Even when temperature fluctuations remain below lethal limits, however, the effects of fluctuating versus constant

16 Page 15 of 35 Canadian Journal of Zoology 15 temperatures can differ (Ashmore and Janzen 2003). Much research has focused on nest site characteristics that influence the incubation environment. Nest site shading by vegetational cover directly influences incubation temperatures (Janzen 1994; Valenzuela 2001; Freedberg et al. 2011). Consequently, females nesting at sites dominated by open canopy often choose shaded nesting areas because of the cooler than average soil temperatures available in such microhabitats (Wilson 1998; St. Juliana et al. 2004). In other populations, females choose nest sites closer to standing water, presumably to maximize cavity moisture in xeric habitats (Morjan 2003). Others have found deep nests to decrease incubation temperatures (Thompson 1988) and moderate temperature fluctuation (Wilhoft et al. 1983; Thompson 1988; Maloney et al. 1990). Wilhoft et al. (1983) and Kaska et al. (1998) even found measurable differences in incubation temperatures and sex ratios between hatchlings from the top and bottom of the same nest, whereas we found a non-significant trend of warmer temperatures in deeper nests, which might be attributable to environmental differences at nest sites. We did not quantify vegetation cover at nest sites because it changes dramatically at our study site as plants grow over the course of incubation. However, the characteristics of nest sites at the time of nesting varied, with some females nesting on open south-facing slopes and others nesting directly under vegetation cover. Because nest temperatures among all groups produced similar average incubation temperatures, it is possible that females adjusted nest depth to the exposure of the nest site and oviposited more deeply at open sites, although much of the variation in maximum incubation temperatures was attributable to depth alone. By providing a mechanism for females to oviposit in deeper soil horizons, subterranean oviposition may allow females to nest in areas unsuitable for shallower nests, which may be important where nest sites

17 Canadian Journal of Zoology Page 16 of are limited or where nest predation rates are high. Females frequently nest near vegetation (Wilson 1998; St. Juliana et al. 2004), but predation rates can also be higher near ecological edges (Temple 1987; Kolbe and Janzen 2002). If dampened temperature fluctuations and lower maximum temperatures of deeper nests allow females to nest farther from vegetated areas, subterranean nesting may allow females to successfully nest in areas with lower predation rates than would otherwise be possible. Finally, females may reduce the risk of attracting predators during the nesting process by being concealed beneath the substrate. Implications for Phenotype Incubation temperature influences a wide variety of phenotypic traits (Booth 2006). Among turtles, incubation temperature may influence hatchling size (Brooks et al. 1991; Du and Ji 2003), post-hatching growth rate (Demuth 2001; Steyermark and Spotila 2001), activity (Janzen 1995), locomotor performance (Janzen 1993; Doody 1999; Du and Ji 2003), and metabolic rate (O Steen and Janzen 1999, Steyermark and Spotila 2000). The reported effects of temperature on these traits varies considerably, however, even among studies of the same species (e.g., O Steen and Janzen 1999; Steyermark and Spotila 2000). Phenotypic traits for which temperature effects are highly consistent across many turtle species are incubation duration and mortality, both of which have profound fitness consequences. Incubation duration is particularly relevant to species that inhabit temperate climates, because many species cannot tolerate overwintering in the egg. This is especially true for ornate box turtles in the northern extent of their range, where hatching and burrowing below the nest cavity appears necessary to avoid freezing. One or more of these temperature-sensitive traits may drive selection for the subterranean nesting that we observed.

18 Page 17 of 35 Canadian Journal of Zoology 17 Among the many ways that incubation temperature influences hatchling phenotype (Packard et al. 1991; Booth 2006), its capacity for determining sex is among the most studied and most demonstrably persistent. The proportion of development occurring at a given temperature, as opposed to the duration of exposure to a particular temperature, determines hatchling sex (Georges 1989; Georges et al. 1994), and because developmental rate varies with temperature, the mean of a fluctuating temperature regime may not accurately predict hatchling sex ratios. Due to the proportionally greater amount of development that occurs at higher incubation temperatures in a fluctuating regime, large daily fluctuations can skew clutch sex ratios toward the sex that is expected at warmer incubation temperatures (Neuwald and Valenzuela 2011), which in this case is female. Given that increased thermal variance is one of the expected outcomes associated with climate change (Fischer and Schar 2009), behaviors that moderate fluctuations may be important to limiting the effects that these changes have on species with temperature-dependent sex determination (Telemeco et al. 2009; but see Refsnider et al. 2013). Nest Outcomes Our observations of nesting behavior results are consistent with previous reports that females may spend many nights searching for nest sites, and may construct preliminary cavities on multiple nights before oviposition occurs (Legler 1960). Sometimes preliminary cavities were constructed large distances (> 100 m) apart, but were more commonly constructed in closer proximity to one another. We documented nesting activity up to nine nights before oviposition. Most often, this behavior resulted in a cavity that was abandoned before completion but sometimes included nests that were complete but lacked eggs. Actual nesting behavior could

19 Canadian Journal of Zoology Page 18 of often only be differentiated from pre-nesting behavior by verifying the presence of eggs. The cumulative hatching rate in our population (83%) exceeded the 42 58% reported in a Wisconsin population (Doroff and Keith 1990). However, we took greater steps to ensure nest success, including caging all of the nests we found, and adding water to nests in 2012 to reduce desiccation. Based upon frequently observed attempts at nest predation, we conclude that caging the nests had a substantial impact on nest survival. The effects of nest watering, on the other hand, were not clear, as hatching success was generally high in both 2011 and 2012 (80% and 87%, respectively), and water supplementation did not occur in Nest watering transiently affected incubation temperatures. Acute declines in temperature of up to 4.0 C (mean = 1.9 ± 0.7 C) were evident in the recorded temperature profiles of some nests after watering occurred, but none persisted longer than 120 minutes and the effects were negligible compared to extended periods of rain or cloud cover. The effect on mean incubation temperatures was imperceptible. Future research on the thermal effects of subterranean nesting should explore the relationship between nesting behavior and canopy cover. Additionally, although temperaturedependent sex determination is well documented in ornate box turtles (Packard et al. 1985; Ewert and Nelson 1991), more detailed data on the pivotal temperature of sex determination are needed for this species to determine the effects that reduced thermal fluctuation may have on hatchling sex ratios. Although we did not find a relationship between maximum incubation temperatures and embryo mortality, others have found that high incubation temperatures and high variation negatively affect hatchling phenotypes and fitness (Mullins and Janzen 2006). In this study population, subterranean oviposition reduced temperature fluctuation and could potentially allow females to compensate for increases in climatic variability that are expected to occur with the further onset of climate change.

20 Page 19 of 35 Canadian Journal of Zoology 19 Acknowledgements We thank the Upper Mississippi River National Wildlife and Fish Refuge, NASA Missouri Space Consortium, and Missouri State University for project funding and support. Thomas Radzio and Eric Tomasovic assisted with erecting and maintaining radio telemetry towers, and the Iowa State University Turtle Camp group assisted with capturing study animals. Denise Thompson provided comments that greatly improved the quality of the manuscript. This project was made possible by a generous equipment loan from the U.S. Army Construction Engineering Research Laboratory. The findings and conclusions in this article are those of the authors and do not necessarily represent the views of the U.S. Fish and Wildlife Service or the U.S. Army Construction Engineering Research Laboratory.

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29 Canadian Journal of Zoology Page 28 of Figure Legends Fig. 1. Nest depths of female ornate box turtles (Terrapene ornata) that were visually confirmed underground (N = 6), those that were confirmed and inferred underground based on comparisons of body length and nest depth (N = 5) and females that were neither confirmed nor inferred to have burrowed on the night of oviposition (nested from the surface; N = 22). Error bars = ±1 SE. Fig. 2. Mean incubation temperatures of ornate box turtle (Terrapene ornata) nests from females that were visually confirmed underground, those that were confirmed and inferred underground (based on comparisons of body length and nest depth) and females that were neither confirmed nor inferred to have burrowed on the night of oviposition (nested from the surface). Error bars = ±1 SE. Fig. 3. Nest depth and incubation temperature of ornate box turtle (Terrapene ornata) nests in order of ascending nest depth. Boxes indicate median, 25 th and 75 th percentile values, and whiskers indicate 10 th and 90 th percentile values. White boxes represent nests from females that oviposited from the surface. Light gray boxes represent nests from females that were inferred to oviposit underground based upon comparisons of nest depth and body length. Dark gray boxes represent nests from females that were visually confirmed to nest underground. Note that the x-axis scale is categorical rather than linear. Fig. 4. Temperature profiles for two ornate box turtle (Terrapene ornata) nests in 2011 that experienced similar mean incubation temperatures but different amounts of temperature fluctuation. Female 14 oviposited underground and had a mid-clutch depth of 16.8 cm and a mean incubation temperature of 28.7 C. Female 1 oviposited from the surface and had a mid-clutch nest depth of 8.4 cm and a mean incubation temperature of