Sex Ratio of Green Turtle (Chelonia mydas) Hatchlings at Sugözü, Turkey: Higher Accuracy with Pivotal Incubation Duration
Abstract
The majority of green turtle (Chelonia mydas) nests in the Mediterranean basin are found on the eastern Mediterranean beaches of Turkey. There are relatively few studies on the sex ratio of green turtle hatchlings for Turkey. In this study, 12 nests on the Sugözü beaches were equipped with temperature data-loggers during the nesting seasons of 2008 and 2009. Temperatures for the entire incubation period and for the middle third were close to the pivotal temperature around 29.0°C. The average incubation duration for nests with known temperature was 52.8 d. An assessment of incubation durations together with histological analysis of 188 gonad samples collected from 103 nests revealed a pivotal incubation duration of 54.3 d and constant temperature equivalent conversion revealed 54 d. This value was identified for the first time for green turtles on the beaches of the eastern Mediterranean in Turkey based on 196 nests with known incubation duration; these nests showed a female-biased sex ratio in 2008 and 2009 (with 54.9% and 56.5%, respectively). Using incubation duration can increase sample size and therefore accuracy of sex ratio predictions without any hatchling sacrifice.
Sex determination is the development of testicles or ovaries from undifferentiated gonads (Hayes 1998). Sex is determined by temperature in sea turtles (Yntema 1979; Yntema and Mrosovsky 1980; Miller and Limpus 1981), including the green turtle (Chelonia mydas Linnaeus 1758), which have a single transition zone (Type I A) in which female hatchlings develop at higher temperatures and male hatchlings at lower temperatures (Bull 1980; Ewert and Nelson 1991). The developmental temperature for which the sex ratio of hatchlings is 1:1 is called the pivotal temperature (Yntema and Mrosovsky 1980; Miller and Limpus 1981), and this value is approximately 29°C for Atlantic populations of sea turtles (Mrosovsky 1994; Ackerman 1997).
The period of sex determination during incubation is termed the thermo-sensitive period, and the duration of incubation shortens as incubation temperatures increase. A 1°C increase shortens the period of incubation by 5 d on average (Mrosovsky and Yntema 1980). Also, metabolic heating plays an important role in the factors affecting the temperature of the nest (Godfrey 1997; Broderick et al. 2001). In addition, it is known that intrabeach thermal variations affect nest temperature and hence the sex ratio (Broderick et al. 2001); however, incubation duration is directly affected by temperature and hatchling emergence is variable under natural conditions (Godfrey and Mrosovsky 1997).
Identification of nest temperature profiles (Spotila et al. 1987; Kaska et al. 1998), monitoring of incubation period (Mrosovsky et al. 1999; Broderick et al. 2000), and identification of hatchling sex can be useful to elucidate population sex ratios and related pivotal values. Identification of hatchling sex ratios is problematic because raising hatchlings in the laboratory or dissection of hatchlings would be unethical. Also, using data-loggers to monitor nest temperatures for a representative number of nests can be costly. In this article, we report green turtle hatchling sex ratios based on histological examination of gonads and incubation durations and temperatures of nests. Our histological results verified that sex ratio estimation is accurate and representative for seasonal assessment with pivotal incubation duration.
METHODS
Study Site
This study was conducted during the nesting seasons of 2008 and 2009 on the Sugözü beaches, Adana (36°48.677′N–35°51.068′E, 36°52.795′N–35°56.017′E), a nesting site for green turtles, which consists of 4 subsections located on the eastern Mediterranean coast of Turkey (Fig. 1). The beaches and surrounding area are subject to intensive human activity in terms of industry and maritime traffic.
Citation: Chelonian Conservation and Biology 15, 1; 10.2744/CCB-1132.1
Collection and Assessment of Data
Daily work was conducted from June to September during both study seasons. Incubation duration (number of days from the date on which the nest was built to the date of first hatchling emergence) was identified for all nests. Temperature data collected from 7 randomly selected nests in 2008 and 5 in 2009 were assessed as “average incubation temperature” (IP) for the value throughout incubation duration, and as “average middle third temperature” (MT) for the value that includes the middle third of the period of incubation, by dividing the incubation duration equally as trimesters. Nest temperatures were measured at 60-min intervals using data-loggers (Gemini Data Loggers-Tinytalk H −30°C/+50°C Part No: TK-0040, UK, with ≤ 0.05°C accuracy) placed (after turtles laid 50 eggs) into the center of the active nest. Incubation durations were identified for other nests in both seasons.
The sex ratio has been estimated using the conversion of total incubation length for nests in the field into the constant equivalent temperature using a modified procedure (based on Mrosovsky et al. 2009) linking sex ratio and incubation duration. In short, the rate of embryonic development is dependent on the incubation temperature and is used as a proxy of sex ratio.
The population of C. mydas closest to Mediterranean populations that has been studied at constant temperature is from the eastern Atlantic, in Suriname (Godfrey and Mrosovsky 2006). These data were used for constant temperature equivalent (CTE) conversion. The CTE is designed to be a measure of fluctuating temperatures that will enable the rate of biological processes in the field to be directly compared with the rate observed in laboratory experiments at constant temperatures (Orchard 1976). It should be noted that the only other green turtle population studied at constant temperatures is from Australia (Miller and Limpus 1981).
The relationship between sex ratio and total incubation duration from the eggs incubated at constant temperatures has been fitted using several sigmoid functions using the R package “embryogrowth” (Girondot and Kaska 2014). The tested functions were logistic (Girondot 1999), Hill and Richards (Godfrey et al. 2003; Hulin et al. 2009), and a newly developed function equivalent to Richards (1959) but with different parameters describing the transition from all-male to pivotal and pivotal to all-female sex ratio. This function is less constrained than the classical Richards (1959) and its modification by Hulin et al. (2009). A model selection was performed using Akaike Information Criterion (AIC) and Akaike weight. In short, the model with the lowest AIC is the model that fit the data well without overparameterization (Burnham and Anderson 2002), and the Akaike weights gave the relative support for each tested model.
The incubation period for natural nests includes the embryonic development and also the time between pipping and emergence from the sand. For C. mydas, this time has been estimated in 2 independent studies as being > 4 d (Hendrickson 1958) and 7 d (Carr and Ogren 1960). We choose a value of 6 d, which is comparable to what is known for other marine turtles (Godfrey and Mrosovsky 1997). This value was subtracted from the incubation duration of natural nests to make the incubation length for these natural nests comparable to laboratory-incubated eggs. Then the sex ratios for these field-incubated nests were estimated from the previous fitted relationship between incubation period and sex ratio. This direct methodology is more satisfactory from a statistical point of view than the original one (Mrosovsky et al. 1999), which linked 1) sex ratio and constant temperature, and 2) constant temperature and incubation period.
Raw data from Godfrey and Mrosovsky (2006) were kindly shared by Matthew Godfrey. R and MINITAB (95% CI, Minitab® Statistical Software v.16) were used for statistical analysis and preparation of graphics.
Collection of Gonads and Histological Analysis
The nests were excavated 3–4 d after last emergence of the hatchlings. The gonads of dead hatchlings and late embryos found in nest during posthatching nest excavation were removed by dissection. In addition, gonads of hatchlings that left the nest but died before reaching the sea and that were located during daily fieldwork by their hatchling tracks were also sampled. The gonads were subject to routine histological preparation: 4–5-μm sections were taken and stained in hematoxylin–eosin. The samples that underwent histological examination were identified as ovaries or testes using the criteria of Yntema and Mrosovsky (1980).
RESULTS
Nest Temperatures
Incubation duration and nest temperature data are summarized in Table 1. Both average IP and MT temperatures of 2009 were higher than those of 2008. Also, average MT temperatures were slightly lower than average IP temperatures for both seasons.
Kaska et al. (1998) found “Sex ratio (% female) = −492 + 18.7 × IP temperature”, and “Sex ratio (% female) = −482 + 18.2 × MT temperature”. Using both equations, green turtle hatchlings from these beaches have a female-biased sex ratio (67.1% IP and 58.5% MT) when both seasons were combined. Sex ratios estimated by IP temperatures in 2009 were higher than those of 2008 but not significantly so (t = −1.73, p > 0.05); however, the ratios estimated by MT temperatures in 2009 were significantly higher than those in 2008 (t = −2.26, p ≤ 0.05).
Incubation Duration
Incubation durations for nests equipped with temperature data-loggers (n = 12) on the Sugözü beaches ranged from 51 to 59 d (mean = 54.6 d) in 2008, and from 47 to 53 d (mean = 50 d) in 2009. The average MT temperature difference for these seasons was 1°C, and the difference in incubation duration was 4.6 d (Table 1).
A negative-biased correlation was found between the IP and MT temperatures, with incubation durations of the 12 sampled nests (ρ = −0.755, p < 0.05 and ρ = −0.730, p < 0.05, respectively). Regression showed a strong relationship both between the IP temperature and incubation duration, and between the MT and incubation duration (r2 = 0.53, p < 0.05; r2 = 0.49, p < 0.05, respectively).
Average incubation duration of all nests was 53.3 d in the nesting season of 2008 (n = 76) and 53.0 d in the season of 2009 (n = 120). Incubation duration was negatively correlated with nesting date (ρ = −0.452, p < 0.001; r2 = 0.20, p < 0.001; Fig. 2).
Citation: Chelonian Conservation and Biology 15, 1; 10.2744/CCB-1132.1
Histological Analysis of Gonads
One hundred eighty-eight gonads were examined from dead hatchlings and late embryos from nests of known incubation duration. Histological analysis revealed an overall female-biased sex ratio of 56.4% (Table 2). As depicted in Table 2, there is a strong relationship between sex ratio and incubation duration between 45 and 64 d (r2 = 0.86, p < 0.001). No males were found (n = 17, %female = 100) in nests with incubation durations ≤ 47 d, and low numbers of females were found (n = 26, %female = 11.5) in nests with incubation durations ≥ 60 d. The ratio close to 1:1 between sexes was found for incubation durations ranging from 51 to 57 d (Table 2).
A clear correlation was found between nest temperature and incubation duration throughout IP and MT periods (Fig. 3). As can be seen in both graphs, the incubation duration corresponding to the pivotal temperature of 29°C is 54.6–55.5 d.
Citation: Chelonian Conservation and Biology 15, 1; 10.2744/CCB-1132.1
Constant Thermal Equivalent (CTE)
The model with the lowest AIC is the Hill model, but the logistic model performs very similarly, as seen in Table 3 (Akaike weights: 0.37 vs. 0.35); therefore, these models cannot be differentiated and both will be used hereafter.
The relationship between sex ratio and incubation duration at constant temperature is shown in Fig. 4 for the Hill model. The logistic model graph looks very similar (not shown). The sex ratio predictions for the 2 models are shown in Table 4 and are very similar.
Citation: Chelonian Conservation and Biology 15, 1; 10.2744/CCB-1132.1
DISCUSSION
The eastern Mediterranean beaches of Turkey are important nesting areas for green turtles, and the species' status in this region is more critical than that of loggerhead turtles (Casale 2015). Few studies have been conducted on the sex ratio of hatchlings of this species, which will be affected by global warming. Despite multiple studies of loggerhead turtles that have been conducted on the beaches of Turkey (Kaska et al. 1998, 2006; Oz et al. 2004; Ucar et al. 2012), the few published studies about green turtles deal with the identification of sex ratio through beach temperatures (Casale et al. 2000), nest and sand temperatures (Candan and Kolankaya 2014), the temperatures and histology (Kılıç and Candan 2014).
Similar studies conducted on green turtles in the eastern Mediterranean report female-biased sex ratio through temperature measurements made in 5 nests (Kaska et al. 1998) and 18 nests (Broderick et al. 2000) in North Cyprus. The studies conducted on Akyatan Beach (Casale et al. 2000) and Sugözü beaches (Candan and Kolankaya 2014; Kılıç and Candan 2014) in Turkey revealed female-biased sex ratios.
The pivotal temperature is approximately 29°C for sea turtles (Godfrey and Mrosovsky 2006). The pivotal temperature for sea turtles in Turkey is estimated at 28.9°C, close to the values found in the current study (Kaska et al. 1998). The temperatures measured and pivotal incubation duration of 54 d, found by CTE for both the IP and MT in this study, are above these values for both seasons. An analysis of the nest temperatures yields a prediction for female-biased hatchling development, although it is not very highly skewed. A strong relationship was found between nest temperature and incubation period, with a shortening of 4.6 d for a 1°C rise in temperature, close to the value found in a laboratory-based study (Mrosovsky and Yntema 1980). A negative relationship was found between egg-laying date and incubation duration, as found previously on the beaches of North Cyprus (Broderick et al. 2000).
The sex ratio of samples taken from nests that emerged from 51 to 57 d is 45.7% female, which is close to 1:1. Nest 2 and Nest 9 had only a 0.1°C difference between MT temperatures but a 9-d difference between their incubation durations, and Nests 3 and 5 also had a 0.1°C difference between MT temperatures but a 4-d difference (Table 1). Although the MT temperatures measured by Kaska et al. (1998) and Broderick et al. (2000) in the nests of green turtles had same average temperature (30.9°C), there is a 8.1-d difference in average incubation duration (58.2 and 50.1 d on average, respectively). Therefore, the possibility of differences between populations (Chevalier et al. 1999) and emergence times (Godfrey and Mrosovsky 1997) should be taken into consideration. Variations (up to 8 d) in emergence should be taken into account in predictions made around the pivotal incubation duration because of effects such as variability in the duration of hatching, hatchling locomotor activity, weather conditions, and metabolic heating (Mrosovsky et al. 2009).
The incubation duration was found to be more highly correlated with overall IP temperature than it is with MT temperature. Metabolic heating that affects the nest temperature increases in the second half of embryonic development, and in the last third in particular (Godfrey et al. 1997; Kaska et al. 1998; Broderick et al. 2001; DeGregorio and Williard 2011). Metabolic heating also affects the sex ratio (Zbinden et al. 2006). Thus, the significant difference of incubation durations of the nests with similar MT temperatures suggests that metabolic heating could be effective for incubation durations. Therefore, a prediction of the sex ratio made by adding the metabolic heating value calculated for a beach to the temperatures of other nests on the same nesting site yields closer predictions (Broderick et al. 2001).
Pivotal incubation durations were found to be 54 d with CTE calculation from raw data of Godfrey and Mrosovsky (2006). Our findings were close to this result: 54.3 d with histological examination and 54.6 d with nest temperatures. Also, although the average incubation duration of nests in 2008 was 53.3 d (n = 76), the incubation duration of nests with temperature data was 54.6 d (n = 7). Likewise, although the average incubation period of nests in 2009 was 53 d (n = 120), the average incubation period of nests with temperature data was 50.0 d (n = 5). If the pivotal incubation duration identified in this study (54.3 d) were used in sex ratio estimation for all nests with known temperature, predictions would be 48% female in 2008 and 72% female in 2009, which does not accurately represent the season; instead, calculation of the average incubation duration of all nests in these seasons, revealed a sex ratio of 54.9% female for 2008 and 56.5% female for 2009. Sample sizes required to represent the accurate incubation duration for 2008 and 2009 were 50 (SD = 2.6367, ME = 0.6456) and 71 (SD = 2.4494, ME = 0.6456) nests, respectively. Moreover, sex ratio prediction based on Hill and logistic equations for field-incubated nests were more highly female-biased than both estimation via incubation duration and histological examination. These differences should be taken into consideration for further studies of CTE calculations.
Increasing sample size would yield more accurate results. Even though use of nest temperatures for sex ratio estimation is commonly employed, inadequate sampling may hinder accurate predictions (Mrosovsky et al. 2009). Where lethal sampling is not appropriate, the number of dead hatchlings obtained from sampled nests may differ, and it is possible to fail to reach the number necessary to make an accurate assessment: a sample size of approximately 20 gonads from each sampled nest is required for an accurate prediction based on incubation temperature (Mrosovsky et al. 2009). It could be possible to increase the histological sample size by grouping the nests according to the incubation durations; therefore, predictions based on histological analysis, measurement of nest temperature, and incubation duration together can lead to accurate calculations.
The Sugözü beaches of Turkey, with 4 subsections (1 = Akkum; 2 = Sugözü; 3 = Botaş; 4 = Hollanda).
Chelonia mydas incubation durations and nesting dates of 2008 and 2009 nesting seasons, Sugözü beaches, Turkey.
Duration of Chelonia mydas incubation, and mean nest temperature of IP (average incubation temperature; +) and MT (average temperature during the middle-third period of incubation; ○) with regression lines (straight line for IP and dashed line for MT).
Relationship between sex ratio and incubation duration for Chelonia mydas eggs incubated at constant temperatures (Godfrey and Mrosovsky 2006).
Contributor Notes
Handling Editor: Jeffrey A. Seminoff
