The Effects of Nest Date and Placement of Eggs in Burrows on Sex Ratios and Potential Survival of Hatchling Desert Tortoises, Gopherus agassizii
ABSTRACT
We studied the effects of timing and placement of eggs by gravid desert tortoises, Gopherus agassizii, on sex ratios and potential survival of hatchlings. We monitored nest placement by female tortoises under seminatural conditions at the Fort Irwin Study Site (FISS) in the Mojave Desert, San Bernardino County, California, United States, and under wild conditions in habitat surrounding FISS. We located 16 nests and found no significant difference between nest placement in the seminatural enclosure at FISS and nest placement in the wild. Gravid females deposited their eggs down the tunnel a mean distance of 0.7 m and buried their eggs at a depth of 8–10 cm from the soil surface. Utilizing this preliminary nesting data, we set up a manipulative experiment to further characterize the effect of nest site and date on sex ratios and survivorship of hatchlings. We constructed 14 egg nests and 9 pseudo-nests in the FISS enclosure and monitored incubation temperatures and resulting hatchling sex ratios. Forty-seven hatchlings emerged (79% survivorship) and 33 were positively sexed. Nests placed early in the reproductive season produced 6 all-female nests and nests placed late in the season produced 4 all-male nests. The mean incubation period was 90 days, which we divided into 3 time periods. Early nests were significantly cooler than late nests during the first and second time period (days 0–30 and days 31–60) and significantly warmer during the third time period (day 61–90). The shallowest pseudo-nest, located 0.2 m down the burrow tunnel, spent a significantly greater time above the critical temperature of 35.3°C than did the 0.4 m pseudo-nest or the egg nest. We hypothesize from these findings that females at the FISS site and surrounding area are selecting distances down the burrow tunnel to lay their eggs that increase the embryos' chances of survival. We found that the proportion of temperature observations above the pivotal temperature (35.3°C) during days 15–45 was a better predictor of hatchling sex than the proportion measured during days 30–61. Using nest date or the proportion of temperature observations above the pivotal temperature as predictors of hatchling sex ratios may be possible for desert tortoises in the Central Mojave Desert.
The conditions under which turtle eggs are incubated have been shown to influence hatchling activity (Janzen 1995), size at hatching (Spotila et al. 1994), and the future growth, physiology, and survival of hatchlings (e.g., Leshem and Dmi'el 1986; Cagle et al. 1993; McKnight and Gutzke 1993; Spotila et al. 1994; Bilinski et al. 2001). Specifically, microhabitat characteristics surrounding the nest have been shown to influence the thermal environment at which eggs are incubated, and subsequently, hatchling survival and the resulting sex ratio in turtles that exhibit temperature-dependent sex determination (TSD; Bull and Vogt 1979; Schwarzkopf and Brooks 1987; Janzen 1994). In addition, the depth at which females lay eggs also has been shown to influence the thermal environment of the nests (Burger 1976; Valenzuela 2001). Because nest depth is correlated with nest temperature, deeper nests have lower temperatures and experience less temperature fluctuations than do shallower nests (Ehrenfeld 1979; Wilson 1998).
The reproductive ecology of desert tortoises in the wild, specifically their nesting ecology, remains largely unexamined (Rostal et al. 1994; Mueller et al. 1998; Meyer et al. 2000). Prior to this study, no research has been published on the effects of timing and placement of eggs on sex ratios and potential survival of hatchling desert tortoises. The lack of definitive research on the subject may be in part due to some inherent challenges to gathering the necessary data. Desert tortoise nests are cryptic and prone to high levels of predation (Roberson et al. 1985; Bjurlin 2001). To better understand the effects of timing and placement of eggs on sex ratios and potential survival of hatchling desert tortoises, we addressed the following questions: 1) Does location of the nest within the burrow tunnel have an effect on hatching success?, 2) Does orientation of the burrow mouth outward have an effect on the resultant sex ratios of hatchling?, 3) Does date of nest placement, early or late in the reproductive season, have an effect on sex ratios of hatchling desert tortoises?, and 4) Can we use temperature data collected from the nest during the incubation period to predict hatchling sex ratios?
METHODS
Study Animal
Populations of desert tortoises, Gopherus agassizii, found west of the Colorado River and North of the Grand Canyon, were federally listed by the US Fish and Wildlife Service as a threatened species in 1990 (US Fish and Wildlife Service 1990). The desert tortoise is a large, herbivorous, terrestrial species that spends most of its time below ground inside burrows that it digs or that other tortoises have dug. Desert tortoises are oviparous and depending on location and habitat may lay from 1 to 3 clutches of eggs annually. Most nesting occurs between May and July with the incubation period lasting 90–120 days (Ernst et al. 1994; Bjurlin 2001). In the Mojave Desert, desert tortoises predominately place their nests within the tunnel of the burrow (Roberson et al. 1985; Turner et al. 1986; Bjurlin 2001). An incubation experiment determined that desert tortoise embryos undergo temperature-dependent sex determination; males were produced between 26.0° and 30.6°C, while females were produced between 32.8° and 35.3°C (Spotila et al. 1994). The pivotal temperature at which the sex ratio is 50:50 was estimated to be 31.8°C (Spotila et al. 1994). Although it is unknown for desert tortoises at what interval of the incubation period sex is determined, other studies have isolated the second third of the incubation period as the interval of sex determination in many turtle species (Mrosovsky and Pieau 1991; Zug et al. 2001; Valenzuela et al. 2003; Doody et al. 2004).
Study Area
Our study took place at the Fort Irwin Study Site (FISS) of the United States Army's National Training Center, San Bernardino County, California (35°06′49″N, 116°29′27″W, 650 m elev.) and the adjacent 5 km2 area of Mojave desert. FISS is located on the southern edge of the National Training Center and is protected from most types of disturbance. The area of desert adjacent to FISS included in our study extends past the southern border of FISS and is open to recreational vehicles and activities. Vegetation at our site consists mainly of creosote bush (Larrea tridentata), burro bush (Ambrosia dumosa), and thorn bush (Lycium pallidum), along with several grasses including Mediterranean annual species (Bromus madritensis ssp. rubens and Schismus barbatus) and galleta (Pleuraphis rigida). FISS rests on a 2% slope of northeast facing hills with sandy soils and occasional dry washes. Two predator-proof enclosures (FISS-I and FISS-II) were constructed in 1990 at FISS as a resource for studying hatchling and juvenile desert tortoises in a seminatural environment (Morafka et al. 1996). Each enclosure contains natural, undisturbed desert vegetation. Since completion of FISS in 1990, gravid, female desert tortoises from surrounding wild populations have been released into the enclosures during the spring and allowed to nest undisturbed. While in the confines of the enclosures, these adult female tortoises dug and utilized many different burrows; thus, natural burrows occur in the enclosures.
Does Placement Site of the Nests by Females Within the Burrow Tunnel Have an Effect on Hatching Success?
In 2001 and 2002, we monitored nest placement by gravid females under seminatural and wild conditions, respectively. In 2001, we captured 6 gravid females within a 1 km radius of FISS and placed them in the FISS enclosures. Females were x-rayed at FISS using a portable x-ray machine (MinXray HF80; 0.08 seconds and 60 kV). At the end of the nesting season, we searched all of the burrows in the enclosures for eggs. When the nest was located within the burrow, we measured the distance from the burrow mouth to the center of the nest using a meter stick.
In 2002, we radio tracked 15 adult, gravid tortoises in the wild throughout the reproductive season. All females were located within 5 km of FISS. Transmitters (AI-2 Holohil Systems Ltd.) were attached to the anterior carapace with adhesive epoxy putty (Devcon®). Weight of transmitters with epoxy was less than 5% of the tortoise's weight. Transmitters were equipped with whip antennas and therefore did not necessitate extra anchoring points along the carapace. A 3-prong yagi antenna was used with an ICOM® RC 10 receiver to locate tortoises. Tortoises were located 3–4 times weekly. With the exception of a weekly weighing, females were not disturbed and handling was minimal. A 100-g minimum weight loss was used as a standard for a tortoise having laid a clutch of 2–3 eggs (Turner et al. 1984). Following weight loss, the nest was located and the distance that the eggs were laid from the burrow mouth was measured. In both 2001 and 2002, we also measured the depth of the egg cavity from the soil surface to the bottom of the eggs.
Using the 2001 and 2002 nest placement data, we set up a manipulative experiment in 2003 in a controlled environment that eliminated the variable of natural predation in the wild. At the beginning of the 2003 reproductive season, we placed the 15 females used during 2002 and an additional 13 adult females captured near FISS into the enclosures. All transmitters were removed, and a small epoxy identification tag was attached to the posterior carapace of each female before they were placed in the enclosures. Following detection of eggs by x-ray, we waited 2 weeks for eggs to fully calcify and then used an injection of oxytocin to stimulate tortoises to lay eggs. Eggs were collected and placed in damp sand until a nesting burrow was prepared. The top of each egg was marked to maintain the embryo's position during placement of the eggs in the nest. We used the collected eggs to construct 14 egg nests from 22 May to 16 July. Each egg nest was composed of 3–5 eggs placed in natural burrows (burrows that had already been dug and used by female tortoises) located in FISS-1. All nest burrows in 2001 and 2002 were located within the canopy margin of vegetation or a geographic feature providing at least partial shade. We selected nest burrows within the enclosure that also met this requirement. Eggs from different females were mixed in each nest to eliminate potential maternal effects. Based on our results of nest site placement in 2001 and 2002, each egg nest was placed between 0.6–0.8 m from the burrow mouth and 8–10 cm to the bottom of the nest cavity. A water-proof data logger (Onset Comp. Inc) was placed in the center of the egg clutch before the eggs were covered to monitor incubation temperatures. Data loggers were programmed to record temperature at 15-minute intervals for the duration of the incubation period.
To compare hatching success of our egg-nest sites to the potential hatching success at other locations within the burrow tunnel not selected by females, we buried data-loggers at pseudo-nest sites and compared temperature data among sites. Because several of the nests constructed by wild females during our 2002 field season were located near the end of the burrow tunnels, we only placed pseudo-nest sites at shallower locations within the burrow tunnel than our egg-nest sites. Pseudo-nests were placed at two distances from the mouth of the burrow, 0.2 and 0.4 m (Fig. 1). Each pseudo-nest consisted of a data logger buried 8–10 cm deep to mimic natural nest depth. Temperature data from pseudo-nests simulated the temperature range eggs would have experienced had they been placed closer to the burrow mouth, without the risk of overheating actual eggs.
Citation: Chelonian Conservation and Biology 7, 1; 10.2744/CCB-0678.1
To determine hatching success in our egg nests, we monitored nesting burrows for emergent hatchlings from August to September 2003. Hardware cloth enclosures were constructed around each nesting burrow to prevent emerging hatchlings from escaping. The enclosures were 0.5 m high with 15 cm of the hardware cloth buried below ground, to prevent hatchlings from burrowing out. Following hatchling emergence, we excavated all nests to look for unhatched eggs. To compare temperature data among egg-nest sites and the two pseudo-nest sites, we divided the average incubation period of 90 days into 3 30-day periods and used a Wilcoxin Rank Sums to compare the distributions of temperature data means among the 3 treatment sites for each 30-day period. When significant differences were found, we used Student's t-test for all pairwise comparisons. Spotila and coworkers (1994) found that incubating hatchlings at a constant temperature of 35.3°C resulted in 72% mortality of embryos. Therefore, we calculated the proportion of temperature data points above 35.3°C for each egg nest and pseudo-nest and compared the distributions of these proportions among the 3 treatments using a Wilcoxin Ranked Sum Test. Our assumption is that nests that spend more time above this critical temperature may experience higher rates of mortality than nests that spend less time above this critical temperature.
Does Orientation of the Burrow Mouth Outward Have an Affect on the Resultant Sex Ratios of Hatchlings?
To determine if burrow orientation has an affect on the resulting sex ratio of hatchlings, we distributed the 14 egg nests that we placed in burrows in 2003 fairly evenly within the 4 compass quadrants: n = 4, 315°–45°; n = 3, 45°–135°; n = 3, 135°–225°; and n = 4, 225°–315°. Upon emergence, each hatchling was given an ID number in waterproof ink on both its plastron and carapace so that we could later match each hatchling to its nest. After hatchlings reached 1 month of age, a blood sample was obtained from each hatchling. The post-carapacial sinus was used for the venipuncture site and approximately 0.2–0.3 mL was collected from each hatchling. Following venipuncture, hatchlings were given water and observed for at least 1 hour for any problems. Each sample was centrifuged for plasma, which was sent to Dr Val Lance, DVM to determine sex using testosterone levels as detailed by Lance (1995). We used G-test of Independence to test if the resulting egg-nest sex ratios were independent of burrow orientation. We only used egg nests that produced all male or all female hatchlings.
Does Date of Nest Placement, Early or Late in the Reproductive Season, Have an Effect on Sex Ratios of Hatchling Desert Tortoises?
In our 2003 experiment, eggs were collected from oxytocin-injected females from 22 May to 16 July. We divided the reproductive season into early and late intervals. The early interval included egg nests that we constructed from 22 May to 2 June, and the late interval included egg nests that we constructed from 17 June to 16 July. We used a Wilcoxin Rank Sums to test for differences in the distributions of nest temperature means for early and late nests. We report the resulting sex ratios from egg nests that developed during these 2 time intervals.
Can Nest Temperature Data be Used to Predict Sex Ratios of Hatchlings?
Although it has been shown that the timing of irreversible sex determination (temperature sensitive period [TSP]) for reptiles with TSD has been isolated to the middle third of embryonic development (Janzen and Paukstis 1991; Mrosovsky and Pieau 1991), most of the field research on turtle species with TSD has focused primarily on aquatic and semi-aquatic species that typically place their eggs near water (e.g., Vogt and Bull 1984; Janzen 1994; Valenzuela 2001; Kolbe and Janzen 2002; Morjan 2003). Because of temperature extremes in the Mojave Desert, desert tortoise nests may begin embryonic development earlier in the incubation period, possibly early enough to shift the TSP to a time before the middle third of incubation. Therefore, we compared temperature data collected during the middle third of incubation (days 30–60) to data collected at an earlier time period (days 15–45). Using a G-test of independence, we compared mean number of temperature data points that were above the pivotal temperature of 31.8°C for egg nests during each time period.
RESULTS
Nest Site Placement and Hatching Success
In 2001 and 2002, we found that gravid females deposited their eggs down the burrow tunnel a mean distance of 0.7 ± 0.08 m (n = 6; range 0.6–0.8) within the FISS enclosure and a mean distance of 0.7 ± 0.15 m (n = 10; range 0.6–1.1) in the wild. We found no significant difference in these distances (df = 1; F = 0.056; p = 0.82). We found no relationship between the distance that a female placed her nest within the burrow tunnel and the total length of the burrow (df = 1; F = 1.79; p = 0.22). Females buried their eggs a depth of 8–10 cm from the soil surface (n = 16).
Of our 28 females that we placed in the enclosures in 2003, 22 females were gravid. These females oviposited a total of 59 eggs. The mean number of eggs oviposited by females was 3.9 ± 0.8 (range 2–6 eggs). Hatchlings began emerging from the 14 egg nests in early August with the last hatchling observed in late September. Of the 59 eggs, 47 eggs hatched (79% survivorship) and 12 eggs were found in the nest cavities unhatched (21% mortality).
Pseudo-nests were placed (at 0.2 and 0.4 m) in 9 of the 14 egg-nest burrows. During the first period (days 1–30), temperatures were significantly cooler for egg nests than they were for the 0.2 m pseudo-nests (χ2 = 6.68, df = 2, p = 0.035). There was no significant difference in temperatures between the egg nests and the 0.4 m pseudo-nests or between the 0.2 m and 0.4 m pseudo-nest during this period. During the middle period (days 30–60), temperatures were significantly cooler for egg nests than they were for both pseudo-nest locations (χ2 = 9.5, df = 2, p = 0.009). There were no significant differences in temperature between the 0.2 m and 0.4 m pseudo-nests. During the final period (days 60–90), there were no significant differences in temperatures among the egg nests and pseudo-nests (χ2 = 0.46, df = 2, p = 0.80).
We found that the proportion of 15 minute temperature data points above the critical temperature of 35.3°C was significantly greater for the 0.2 m pseudo nests than for the 0.4 m pseudo-nests and the egg nests (χ2 = 15.6, df = 2, p = 0.0004). The proportion of 15 minute temperature recordings above the critical temperature of 35.3°C did not differ significantly between the 0.4 m pseudo-nests and the egg nests (Table 1).
Orientation and Sex Ratios
A total of 33 hatchlings were positively sexed. The remaining 14 hatchlings that survived escaped the hardware cloth pens before they could be marked; therefore, we were unable to assign these hatchlings to an egg nest. Ten egg nests produced either all male or all female hatchlings (all male: n = 4; all female: n = 6). Because our resulting sample sizes of nests in each of the 4 compass quadrants were small, we divided burrow orientations into 2 compass quadrants: east facing (0°–180°) and west facing (181°–360°) directions. Using data logger temperature data, we found no differences in distributions of mean temperatures between east and west facing burrows during any of the 30 day time periods (days 0–30: χ2 = 0.92, df = 1, p = 0.34; days 31–60: χ2 = 1.64, df = 1, p = 0.20; days 61–90: χ2 = 0.10, df = 1, p = 0.75). We found that hatchling sex ratios were independent of burrow orientation (G = 0.66, df = 1, p > 0.5).
Timing of Nest Placement and Sex Ratios
During the 2003 reproductive season, we constructed 7 egg nests in the early interval (22 May–2 June) and 7 egg nests in the late interval (17 June–16 July). Two of these nests were not used in our analyses because one produced no hatchlings, and the other produced only one hatchling. Early nests (n = 6) produced 4 all-male nests and 2 mixed nests; late nests (n = 6) produced 6 all-female nests. Of the 33 hatchlings that we were able to sex, early nests produced 13 males and 4 females and late nest produced 0 males and 16 females (Table 2). Because numbers of male and female hatchlings are not independent, we were unable to test for difference in the sex ratio between treatments. We found that early nests were significantly cooler than late nests during the first and second time periods (days 0–30: χ2 = 8.3, df = 1, p = 0.004; days 31–60: χ2 = 3.7, df = 1, p = 0.05) and were significantly hotter than late nests during the third time period (days 61–90: χ2 = 6.6, df = 1, p = 0.01; Table 3).
Temperature Data and Sex Ratios
Temperature data for 30-day time periods days 15–45 and 30–60, produced close to 2880 15-minute interval data points for each nest. The proportion of temperature data points above the pivotal temperature of 31.8°C for each nest, and the hatchling sex ratios are summarized in Table 2. During both time periods, nests that produced all female hatchlings (n = 6) spent a greater mean number of temperature data points above the pivotal temperature of 31.8°C than did those that produced all male hatchlings (n = 4) (days 15–45: G = 3625, df = 1, p < 0.001; days 30–60: G = 562, df = 1, p < 0.001). Nests that produced all female hatchlings spent a greater mean number of temperature data points above the pivotal temperature during time period 15–45 than time period 30–60 (G = 52, df = 1, p < 0.001; Table 2).
DISCUSSION
Nest Site Placement and Hatching Success
During the 2001 and 2002 field seasons, we found that adult females deposited their eggs between 0.6 and 0.8 m down the burrow tunnel. Although many of the nest burrows observed during both years extended in length beyond the placement of the eggs, we found no relationship between nest placement and the total length of the burrow. Therefore, it appears that desert tortoises in our study may be selecting a particular distance down the burrow tunnel to place their eggs rather than just laying them at the end of the tunnel.
Many investigators have shown that the depth at which female turtles lay their eggs from the surface of the ground and the microhabitat surrounding the nests influence the thermal environment of the nests (e.g., Burger 1976; Janzen 1994; Wilson 1998; Bjurlin 2001; Valenzuela 2001). In general, these studies found that relatively deep nests with less solar exposure experienced cooler overall incubation temperatures while shallow nests with greater solar exposure experienced warmer incubation temperatures. Deeper nests also experience less temperature fluctuations throughout the incubation period (Ewert 1979). Several studies have documented females selecting nest sites with microhabitat characteristics that have been shown to produce specific thermal conditions during incubation, and that these nest sites differ from random sites (Wilson 1998; Kolbe and Janzen 2002; Morjan 2003). The depth and solar exposure of a nest site are 2 factors that females can select that may increase the potential success of their reproductive effort.
Some desert tortoises have been shown, at other locations in the Mojave Desert, to place eggs on the burrow apron (Meyer et al. 2000; Bjurlin 2001). We predict that at our study site, eggs placed at shallower locations within the burrow tunnel would not have survived because of extremely hot temperatures. Laboratory experiments have shown that desert tortoise eggs incubated at a constant temperature of 35.3°C resulted in 72% embryo mortality (Spotila et al. 1994). We found that our egg nests were significantly cooler than the pseudo-nests during the first 60 days of incubation. Of our 17 pseudo-nests, 5 during incubation days 0–30, 17 during days 31–60, and 9 during days 61–90 reached temperatures above the critical temperature of 35.3°C; whereas, of our 9 egg nests, 2 during incubation days 0–30, 2 during days 31–60, and 2 during days 61–90 reached temperatures above the critical temperature. We hypothesize that at our study site female desert tortoises may be selecting distances down the tunnel to lay their eggs that increases the embryos' survival probability.
Orientation and Sex Ratios
East-facing burrow openings receive a predominance of early to midmorning sun and less of the warmer afternoon sun. West-facing burrows receive the opposite treatment, very little morning sun, and a much greater amount of afternoon sun. The amount of directional solar exposure a nest receives has been documented to affect sex ratios in Chrysemys picta bellii (Janzen 1994), who found that nests with less southern exposure were cooler and male-biased. In our study, we found that mean temperatures of our egg nests placed at 0.6–0.8 m down the burrow tunnel did not differ between east- and west-facing burrows during any of the 3 30-day time periods. Also we found that hatchling sex ratios were independent of burrow orientation. Timing of nest placement, either early or late in the reproductive season, may have masked any effect of orientation on hatchling sex ratios. To determine whether orientation may have an effect on sex ratios, egg nests in a larger sample of burrows facing different compass directions during both the early and late reproductive season would need to be monitored to tease apart the effects of orientation and timing of nest placement.
Timing of Nest Placement and Sex Ratios
During 2003, gravid females in the FISS enclosure experiment oviposited eggs throughout the reproductive season, from 22 May to 16 July. For our experimental nests, early nests experienced relatively cool temperatures and late nests experienced relatively warm temperatures during the temperature sensitive period. Monitoring ambient temperatures, Bjurlin (2001) found that desert tortoises nests laid in the south-central Mojave Desert experienced changing temperature regimes with season. Our early nests produced predominately males, and our late nests produced predominately females. As in our study, nests of the pig-nosed turtle, Carettochelys insculpta, oviposited late in the reproductive season were hotter and produced more females than did those laid early in the reproductive season (Doody et al. 2004). Seasonal changes in temperature may produce predictable differences in incubation temperatures, which in turn may result in seasonal differences in sex ratios (Doody et al. 2004; Harlow 2004).
Using nest date as a predictor of hatchling sex ratios of desert tortoises in the Central Mojave Desert may be possible. Desert tortoise egg nests laid early in the season would produce male-biased hatchling sex ratios because, although they start out warmer than nests of aquatic turtle species, they are relatively cool when compared to nests laid late in the season. Egg nests laid late in the season would produce female-biased hatchling sex ratios because they experience relatively warm temperatures during the first part of incubation and cool temperatures at the end.
Temperature Data and Sex Ratios
Natural incubation conditions are difficult to accurately reproduce in a laboratory setting due to the shifting temperature and humidity regimes typically experienced in nature. Variation in nesting patterns, geographic location, and climate found among and within turtle species with TSD may be too extreme for the application of specific rules. The thermal environment of the nest burrow is likely the determining factor in sex determination for desert tortoises but has not been significantly characterized. In the past, temperature data from nest cavities in other turtle species have been evaluated using mean nest temperatures, but mean nest temperatures have not been shown to be good predictors of hatchling sex ratios (Schwartzkopf and Brooks 1985; Georges 1989; Weisrock and Janzen 1999). Valenzuela (2001) noted that although attempts to use the mean temperature and various alternative models to the mean as a predictor of sex ratios, none had proven to be a good predictor of sex ratios in Podocnemis expansa. The number of hours above a critical temperature during days 29 and 30 of incubation proved to be the best predictors of resultant sex ratios for P. expansa (Valenzuela et al. 1997). The failure of existing models to accurately predict sex ratios for P. expansa suggests that a model's ability to predict sex ratios from nest temperatures may be handicapped by the amount of variation in incubation conditions among species. The development of species-specific models may be necessary for some species such as P. expansa and G. agassizii.
The timing of sex determination or the TSP has been isolated to the second third of the incubation period for many turtle species with TSD (e.g., Bull and Vogt 1981; Yntema and Mrosovsky 1982; Wibbels et al. 1991). Using the middle third rule, sex of hatchlings with an average incubation period of 90 days would be determined during the 30–60 day segment of incubation. However, we found that the proportion of temperature observations above the pivotal temperature (31.8°C) during days 15–45 was a better predictor of hatchling sex ratios than was the proportion of temperature observations above the pivotal temperature during days 30–60. Nests from our study started out warm, unlike nests of aquatic turtle species that are laid in early spring and start out comparatively cool (e.g., Chrysemys picta, Cagle et al. 1993; Caretta caretta, Georges et al. 1994; Kinosternon bauri, Wilson 1998).
We hypothesize that the thermal sensitive period for desert tortoise eggs laid in the Central Mojave Desert may be earlier than the typical middle third period. Using data loggers in nests and recording the number of 15-minute temperature observations above the critical temperature for sex determination in desert tortoises during incubation days 15–45 may be a good predictor of sex ratios; however, additional work needs to be done using data loggers in wild nests and comparing temperature data obtained with resulting sex ratios in wild hatchlings.
CONCLUSIONS
The research presented here has characterized the natural nesting microhabitat of a small population of desert tortoises and investigated how orientation, nest placement, and nest date affect the thermal environment and resultant hatchling sex ratios. Our findings indicate that nest placement within the burrow is an important determinant in what temperature regime the nest experiences during incubation and is, therefore, also a significant factor influencing resultant hatchling sex ratios. Date of nest placement at our study site also appeared to be a good indicator of resultant hatchling sex ratios; early nest produced a greater proportion of males and were comparatively cooler than late nests which were warmer and produced a greater proportion of females. Temperature data collected during days 15–45 appeared to be a better indicator of hatchling sex than temperature data collected during the middle third of incubation, days 30–60, where sex determination has been hypothesized to occur for most species with TSD.
If maternal selection of a nest-site is based on microhabitat characteristics that differ from other available sites, it is imperative for public and private land managers to understand that relationship. Understanding the factors influencing nest success, hatchling survivorship, and hatchling sex ratios is essential to the continued survivorship and health of desert tortoise populations.
Diagram of constructed nests in 2003 with 2 pseudo-nests located 0.2 m and 0.4 m down the burrow tunnel and the egg nest located between 0.6 and 0.8 m.
