RESEARCH ARTICLE

Loggerhead Sea Turtle Environmental Sex Determination: Implications of Moisture and Temperature for Climate Change Based Predictions for Species Survival JEANETTE WYNEKEN* AND ALEXANDRA LOLAVAR Department of Biological Sciences, Florida Atlantic University, Boca Raton, Florida

ABSTRACT

J. Exp. Zool. (Mol. Dev. Evol.) 324B:295–314, 2015

It has been proposed that because marine turtles have environmentally determined sex by incubation temperature, elevated temperatures might skew sex ratios to unsustainable levels, leading to extinction. Elevated temperatures may also reduce availability of suitable nesting sites via sea level rise. Increased tropical storm activity can directly affect nest site moisture, embryonic development, and the probability that nests will survive. Here, we question some of these assumptions and review the limits of sex ratio estimates. Sea turtles may be more resilient to climate change than previously thought, in part because of hitherto unappreciated mechanisms for coping with variable incubation conditions. J. Exp. Zool. (Mol. Dev. Evol.) 323B:295–314, 2015. © 2015 Wiley Periodicals, Inc. How to cite this article: Wyneken J, Lolavar A. 2015. Loggerhead sea turtle environmental sex determination: Implications of moisture and temperature for climate change based predictions for species survival. J. Exp. Zool. (Mol. Dev. Evol.) 323B:295–314.

To test predicted effects of increasing temperature due to climate change, we sampled primary sex ratios from a major Florida loggerhead rookery under typical and more extreme climatic conditions. Neonate turtle sex was verified using gonad and gonadal duct morphology. Samples were 100% female when weather conditions were hotter and drier than normal. Eggs that incubated in hot but wet years produced mixed sex samples, including samples that were strongly male- or female-biased. Experimental clutches were lab-reared under moist conditions at temperatures predicted to produce female-biased sex ratios. They produced
90% males. Taken together, our results suggest a source of resiliency not previously recognized: female-bias under hot dry conditions and male-bias under hot wet conditions. These findings indicate more conditional responses to climate change than previously considered, and cause us to question the generally accepted assumption that changes in temperature, alone, will have a negative impact on sex ratios. Temperature is the major environmental factor driving the direction of sexual differentiation in many reptile species (Bull,

'80; Bull et al., '82; Ewert and Nelson, '91; Valenzuela and Lance, 2004). Recent literature highlights the potential impact of

Grant sponsor: U.S. EPA STAR Program GAD; grant number: R82-9094; grant sponsor: Disney World Conservation Fund/Disney Wildlife Conservation; grant sponsor: Save Our Seas Foundation; grant sponsor: National Save the Sea Turtle Foundation; grant sponsor: AWC Foundation; grant sponsor: Nelligan Sea Turtle Research; grant sponsor: NSF URM; grant number: 0839250. Additional supporting information may be found in the online version of this article at the publisher's web-site  Correspondence to: Jeanette Wyneken, Department of Biological Sciences, SC136, Florida Atlantic University, 777 Glades Rd, Boca Raton, FL 33431-0991. E-mail: [email protected] Received 29 August 2014; Accepted 2 March 2015 DOI: 10.1002/jez.b.22620 Published online in Wiley Online Library (wileyonlinelibrary.com).

© 2015 WILEY PERIODICALS, INC.

296 anthropogenically-driven climate change effects on species with temperature dependent sex determination (TSD), a form of environmental sex determination (ESD). Predicted effects on a variety of reptiles range from increased extinction risk caused by an extreme skewing of sex ratios (Janzen, '94; Miller et al., 2004; Nelson et al., 2004; Mitchell et al., 2008; Pintus et al., 2009; Kallimanis, 2010; Mitchell and Janzen, 2010; Escobedo-Galv
an and Gonz
alez-Salazar, 2012; Woolgar et al., 2013) to more optimistic expectations of resiliency (Warner and Shine, 2008; Hulin et al., 2009; Kallimanis, 2010; Silber et al., 2011; Marcovaldi et al., 2014). The lack of agreement suggests that the quality and scope of available data, as well as the analytical approaches used, may leave much to be desired! This paper reviews and illustrates some of the gaps in previously published studies and provides examples of how more analytical approaches that combine both field and lab data and considers both temperature and moisture, which are important to developing embryos, may lead to a new (and hopefully more accurate) set of predictions. Chelonians are among the reptiles discussed commonly in the climate change literature because many species are imperiled and most species have TSD. The Turtle Taxonomy Working Group (2014) reports that 40.3% of all recognized modern turtle and tortoise species are imperiled (critically endangered, endangered, or vulnerable). These statistics are alarming and point to a substantial need for more robust demographic data, including primary (hatchling) sex ratios. Marine turtles tend to have highly female-biased hatchling productions (Wibbels, 2003). Consequently, there is a concern that incubation conditions will result in the production of too few males for populations to sustain themselves. Primary sex ratios are important because they are the essential demographic baseline that underlies any change in the sex ratios exhibited by older turtles. It is at this life stage that highly skewed sex ratios may be detected—long before reproductive consequences are realized. Primary sex ratio changes are a consequence of how ecological forces direct the development of embryonic sex (i.e., sex determination). In this paper, our goals are to review the assumptions used to estimate primary sex ratios and present new evidence for how the incubation environment drives primary sex ratios of developing sea turtle embryos. Previous studies have focused on thermal effects on turtle sex development and implications of small changes in temperature (Yntema and Mrosovsky '79, '80, '82; Bull, '80; Mrosovsky and Yntema, '80). Yet, in the evolutionary history of turtles, there have been multiple periods of climate change. There is a need for a better understanding of how environmental interactions not necessarily restricted to temperature, affect the sexual differentiation of embryos within the nest. Once those interactions are understood, we will be in a better position to predict how climate change may affect the long-term survival of species with ESD systems. J. Exp. Zool. (Mol. Dev. Evol.)

WYNEKEN AND LOLAVAR

REVIEW OF CHELONIAN SEX RATIO STUDIES AND PREDICTIONS OF CHELONIAN SPECIES SURVIVAL DUE TO CLIMATE CHANGE Among the many imperiled chelonian taxa, marine turtles frequently are deemed particularly vulnerable to changing climate because extant species nest coastally, and because they have a cooler male/warmer female TSD system (e.g., Hawkes et al., 2009; Hulin et al., 2009; Poloczanska et al., 2009; Fuentes et al., 2010). Loggerhead (Caretta caretta) nests from peninsular Florida beaches, for example, appear to be strongly female biased (Hanson et al., '98; Mrosovsky and Provancha, '92; reviewed by Wibbels, 2003). Because males are the rarer sex at most locations, the concern is that with a further increase in temperatures, even fewer males will hatch and sex ratios will become too skewed toward females to support future reproduction. According to this scenario, the density of males could be so low that dispensation (a component of Allee effects) could be expressed within populations (Courchamp et al., 2006). To assess such concerns, we first review the challenges and limitations of measuring primary sex ratios and discuss limits to the evidence that climate change poses a threat to sea turtle sex ratios. The Use of Proxies Empirical measures of primary sex ratios in situ are difficult to obtain and rarely done because hatchling and neonate sea turtles are not sexually dimorphic. As a result, sex ratios usually are estimated from either proxies of sex ratios (Girondot et al., 2010) or proxies of the parameters presumed to direct those ratios and those proxies are rarely tested. Some proxies (Table 1) are less accurate and less powerful statistically than others. To reflect these issues we categorize proxies by how many steps they are removed from the actual embryonic sex ratio. For example, those removed by a single step are categorized as “first level proxies,” those removed by two steps as “second level proxies,” and those removed by three or more steps as “third level proxies.” An increase in proxy “distance” decreases the reliability of the sex ratio estimate. First Level Proxies. First level proxies are small samples of hatchlings from a single nest used to infer sex ratio of the whole nest (Table 1). Obtaining a sufficient sample of hatchlings is a common problem associated with estimating sex ratios. Until recently, sex ratios based upon empirical data depended upon hatchlings that were sacrificed to verify their sex by histology. Sex ratios based upon large samples of sacrificed hatchlings are rare (Godfrey, '97; Mrosovsky and Provancha, '89) because most species are protected. Destructive sampling approaches are ethically challenging and usually restricted or prohibited. Wyneken et al. (2007) developed a reliable technique to determine sex by laparoscopy. However, raising enough hatchlings to a large

ENVIRONMENTAL SEX DETERMINATION IN LOGGERHEAD TURTLES

297

Table 1. Steps in using proxies to estimate hatchling sex ratios. Proxy 1st Level 2nd level

3rd level

Step 1 Identify of sexes of live turtle samples Incubation temperature Dead-in-nest sex ratio Incubation duration Temperature summary metric (Incl. CTE, mean, pivotal, etc.) Collect meteorological or oceanographic data

Step 2

Step 3

Step 4

Sex ratio estimate Estimate middle 1/3 of development (TSP)  Live turtle sex ratio Estimate TSP Estimate middle 1/3 of development (TSP) Estimate sand temperature

Sex ratio estimate Sex ratio estimate Identify thermal conditions of TSP Infer uniform response (no maternal or other modifiers) Estimate incubation temperature

Sex ratio estimate Sex ratio estimate Sex ratio estimate

Using proxies to estimate hatchling sex ratios necessarily requires making assumptions about the relationship of one step to the next. Each step may make the sex ratio estimate less accurate because the data are inferred rather than measured. The examples for each proxy level do not provide an exhaustive list. Note that there likely is a continuum of proxy levels between levels 2 and 3.

enough size (
120 g) for the yolk sac to be absorbed and so there is enough body space to conduct these examinations is expensive, labor intensive, and limited by logistics and regulatory restrictions to small samples, even though the procedure is non-lethal. Yet, large samples are necessary to reliably estimate nest sex ratios because of seasonal and spatial variation in environmental conditions (Godfrey and Mrosovsky, ’97). It is common to statistically “under sample” nests. Samples of 10 or even 20 hatchlings from nests containing
80–100 turtles are “common” among the few studies that collect empirical sex ratio data through these methods (e.g., LeBlanc et al., 2012). Small samples of hatchlings can lead to fallacious sex ratio results because the confidence intervals for hatchlings sampled from nests producing so many more turtles are quite large (Mrosovsky et al., 2009; Spotila et al., '87). Thus, studies that use small sample sizes to estimate nest sex ratios are limited in accuracy of this sex ratio proxy. Adequately sampling sex ratios across an entire rookery is equally challenging because the variance in nest sex ratios over an entire beach is unlikely to be unimodal and, to date, has not been calculated. Procedures to assess statistical power of sampling protocol for multimodal distributions are lacking. Second Level Proxies. Second level proxies include several methods for estimating sex ratios. Dead hatchlings scavenged following a nest emergence are commonly used as a measure of sex ratio. These measures are used because the dead hatchlings are available and are presumed to reflect the outcome of the same incubation environment as the turtles that emerged (Kaska et al., 2006; Wibbels et al., '99). However, they may not be an unbiased sample. Their numbers are often small, and they vary in decomposition state so that a histological examination of the

gonads to identify sex may not be possible (further limiting sample size). As a source of primary sex ratios, they compound both sample size issues and unknown bias because the locations in the clutch that produces dead versus live hatchlings may differ. Only one study (LeBlanc et al., 2012) compared sex ratio results obtained from two different proxies: dead-in-nest hatchlings and 10-hatchling samples of those clutches. They found no correlation between the two sex ratio measures. In this instance, both the dead-in-nest and sacrificed hatchling samples were inadequate and so neither gave consistent results. Another second-level proxy is the use of nest incubation temperature to infer knowledge of the results of gonadal differentiation. Typically, the incubation temperature during the presumed thermosensitive period (TSP) is used. However, the value of incubation temperature without verification of sex ratios (described below) or verification of developmental stages is questionable. Godfrey et al. ('97) sampled leatherback eggs in the field and found variation in stages across the TSP when gonadal development is environmentally directed. In freshwater turtles, incubation proceeds linearly at temperatures that yield optimal developmental rates (Georges et al., 2005); faster developmental rates are associated with warmer temperatures, up to a point (Bowden et al., 2014). At temperatures above some threshold, development slows thus specific developmental stages are difficult to infer without sampling of embryos for stage verification (Girondot et al., 2010). Third Level Proxies. Third level proxies also comprise several types of estimators. These include use of incubation duration to infer knowledge of the middle third of development (TSP) when the incubation environment directs gonadal differentiation (Miller stages 22–26; Miller, '85). Short incubation durations J. Exp. Zool. (Mol. Dev. Evol.)

298 occur when nest temperatures are relatively high and are associated with rapid development; longer durations occur when incubation temperatures are relatively low and development slows. Sex ratios in the nest are assumed to be related to those of laboratory-incubated eggs (i) kept at constant temperatures, or (ii) that completed incubation over the same length of time, or (iii) that have the same average pivotal temperatures so that inferences about field sex ratios can be made. In fact, incubation temperature and other conditions within natural nests such as water potential (Steyermark, '99) can vary periodically during incubation, yet result in an incubation period that is identical in length to a clutch kept in an incubator. If that variation in the nest occurs during the TSP, the two sex ratios (nest vs. incubator) may not be comparable. In summary, the middle third of incubation is easily identified under controlled laboratory conditions of constant temperature and usually reflects the expected developmental stages. The same cannot be so accurately delineated under field conditions. Other characteristics of incubation, such as diel variation in incubation temperatures or time incubating at a particular temperature during the TSP (e.g., Georges, 2013), are used with some success as correlates of sex ratios in freshwater turtles. But such approaches may be less useful for sea turtle sex ratios because their nests are deeper than most freshwater turtles and are thermally buffered from large diel thermal variations. In freshwater turtles, incubation rates proceed roughly linearly across temperatures that allow for optimal developmental rates (Georges et al., 2005). Bowden et al. (2014) summarize studies that show that development slows at the warmest temperatures that allow successful development. Embryonic stage-incubation temperature relationships are difficult to infer without sampling of embryos for stage verification. Consequently, with few exceptions (e.g., Godfrey et al., '97), there is unmeasured error associated with procedures used to assign specific developmental stages (when sex is being determined) based on incubation durations at particular temeperatures (Girondot et al., 2010, Girondot and Kaska 2015). The use of Constant Temperature Equivalent (CTE) attempts to resolve some of the mismatch between laboratory constant temperature results and field results. Conceptually, CTE approaches mathematically transform field incubation temperatures to a constant temperature that would result in the same developmental rate (see Cuong et al., 2008; Georges et al., 2004). The assumptions of the CTE approach are that the developmental rates and the appropriate range and resolution of incubation temperatures are known. Recent use of climatic data to estimate current and past sex ratios are intriguing but are also third level proxies (Hays et al., 2003; Hawkes et al., 2009; Hulin et al., 2009; Poloczanska et al., 2009; Fuentes et al., 2010). Some correlative models use oceanographic temperature data to retrospectively estimate sex ratios, again as third level proxies for nest temperatures (e.g., J. Exp. Zool. (Mol. Dev. Evol.)

WYNEKEN AND LOLAVAR Laloë et al 2014; Fuentes et al., 2009). Some studies include extrapolations of sand temperatures at nest depths to infer incubation temperatures. Other studies used ocean or air temperatures to infer sand temperatures, which are then used to infer nest incubation temperatures, and from those extrapolations calculate sex ratios expressed by the embryos. The inferred incubation temperatures vary among studies, are unspecified and may rely on lab-based values (transitional range of temperature [TRT] that can produce both sexes in a sample of eggs or pivotal temperatures; Mrosovsky and Yntema, ’82; Mrosovsky, '88; Mrosovsky and Pieau,’91) to estimate if clutches produce one or both sexes (Girondot et al., 2010). These extrapolative methods used for climate change predictions may be informative at the thermal extremes that allow successful incubation but are questionable in reality. They effectively ignore important conditions experienced by in situ eggs (for e.g., vegetative cover, proximity of clutches to ground water, tidal amplitude effects on nest temperatures, and storm effects). They necessarily lack spatial or temporal resolution because the historic metrics are recorded at scales appropriate for describing weather and climate but not at scales most developing embryos experience. In our view, the third-level proxy methods, while intriguing, represent questionable procedures for estimating the conditions actually experienced by the embryos inside the nest. The Key Assumption. The key assumption underlying most of these proxies is that temperature is the biologically most important metric to correlate with hatchling sex ratio estimation. Many careful studies show that incubation temperature correlates well with hatchling sex ratios under laboratory conditions, and with sex ratios from natural nests that experience incubation temperatures persisting below or above the TRT throughout the middle third of incubation (Wibbels, 2003). However, debate remains as to which temperature metric is the most appropriate descriptor of incubation conditions that are relevant to developing embryos during the TSP. Mean temperature appears often as a descriptive metric but some question this approach. Godfrey and Mrosovsky (2001) reports that more than 75% of the variation in incubation duration is accounted for by temperature, but the remaining
25% is unexplained. Others have struggled with unexplained variation between sex ratios and mean incubation temperatures (e.g., Bull, '85; Godley et al., 2001). Kaska et al., (2006) had success modeling the relationship of loggerhead sex ratios to mean incubation temperature at Turkish rookeries. Other studies use min and max temperatures, range of temperatures, pivotal temperature, CTE, or days of incubation occurring above or below the pivotal temperature; models vary with study, species, and even vary within species at different locations (e.g., Georges '89, Georges et al., 2004). Extensive review of the thermal proxies is beyond the scope of this paper; excellent reviews published by Girondot et al. (2010)

ENVIRONMENTAL SEX DETERMINATION IN LOGGERHEAD TURTLES and Bowden et al. (2014) discuss those used for freshwater and marine turtles. Nevertheless, the fundamental challenge is that the mechanisms that determine TSD are not fully understood under field conditions. TSD mechanisms acting under laboratory conditions (reviewed by Matsumoto and Crews, 2012) show more certainty but applications of those mechanisms to ecological conditions experienced by developing embryos remains wanting. In the absence of such a mechanistic understanding, selecting the best thermal metric to predict the consequences of climate change on sex ratios of embryos compounds several kinds of uncertainty. Temporal Bias in Sex Ratios An additional problem arises when sex ratio sampling is not done across the nesting season. In a multiyear study of loggerhead primary sex ratios, samples represented the warmer part of the season (Mrosovsky and Provancha, '92). Sampling the part of the season that produces the most hatchlings may capture the predominant sex ratio but may provide a biased estimate of the sex ratio because other parts of season are not included (such hatchlings may have different survival prospects too). The extent of such likely varies geographically, depending upon local climatic conditions (Godfrey and Mrosovsky, ’97). Within the context of the kinds of uncertainties described above, we summarize and critically review the current evidence that climate change poses a threat to marine turtles because of its effect on their predicted sex ratios. Are the Nest Temperature Predictions Meaningful? Nests that receive greater solar exposure are reported to become warmer (Booth and Astill, 2001; Wood et al., 2014). Several studies identified correlation between sand temperatures and air temperatures (e.g., Hays et al., 2003; Fuentes et al., 2009). However, greater solar exposure does not consistently result in warmer sand temperatures because sand materials differ in how much solar energy they reflect (albedo) or absorb. In most instances, it is only the upper few centimeter of the beach that warm diurnally so that if the eggs are placed deeper (the normal condition for marine turtles), they are thermally buffered from large diel fluctuations (as well as seasonal warming, Hays et al., 2001). An extrapolation of effects from solar exposure to the sand and specifically to the incubation environment is tenuous because nests are biophysically different from sand. Each clutch of roughly 100 eggs is closer in properties to water or agar than it is to sand because the eggs are spherical, mineral-bound packets of albumin and yolk. Water accounts for
65–80% of the wet mass of sea turtle eggs (Wallace et al., 2006). Eggs are physiologically dynamic, exchanging gases and water as the embryo develops (Ackerman, '97). A clutch is an egg mass with little or no sand surrounding most of the individual eggs. Only the peripheral eggs are bounded by sand, and that effect is limited to a portion of their surface. Hence, modeling the nest environment by its

299 surrounding sand temperature is at best a crude estimate of the microclimatic conditions experienced by the clutch. Similarly, air temperatures do not account for microhabitat differences that might change sex ratios. Nests in the same region can differ in their microhabitat due to proximity to vegetation and shore or € et al., 2004). ground water (Godfrey and Mrozovsky, ’99; Oz The developing embryos change the nest environment; they release metabolic heat so nests are thermogenic during that latter third of development. Most studies find this effect is pronounced after embryonic sex is determined (Godley et al., 2001; Zbinden, 2006). However, at least two studies suggest metabolic heat could contribute to the incubation environment during the TSP (Broderick et al., 2001; Girondot and Kaska 2015). Microhabitat variations due to other factors such as cloud cover, locally or regionally heavy rainfall that cool nests (Standora and Spotila '85; Wyneken, unpubl. data), and shade from maritime forests, dune vegetation, or buildings (Mrosovsky et al., '95) influence sand temperatures and nest temperatures. In many cases, models that infer incubation temperature from air temperature do so by relying upon meteorological data from stations that usually are remote from the beach (e.g., Fuentes et al., 2009; Matsuzawa et al., 2002). These third level proxies provide correlative estimates of possible incubation temperatures, but they cannot account for microhabitat characteristics, thermal buffering as a consequence of nest depth, patchiness of rainfall or shade, and sand albedo effects on nest temperatures. Where meteorological data (3rd level proxies) are collected away from the beaches, they are even less likely to adequately describe the nest microhabitat features that are critical to determining sex ratios for the reasons described above. Often there are multiple problems with data used for predictions about sex ratios. For example, Godley et al., (2002) associated sex ratios with sand and air temperatures. Those sex ratios were based on sample sizes that were too small to adequately estimate clutch sex ratios (Mrosovsky et al., 2009). Furthermore, the data were obtained for a portion of a single season, and were based upon temperature/sex ratio relationships from different regions and using several species to populate the curve more fully. Hence, it is difficult to know what that relationship means for any one species when the data set consists of segments to create the composite. While these approaches served to develop a field of study in its early stages, later studies should develop and stand on stronger foundations. Yet, the Godley et al. (2002) study serves as the “verification” for several others (e.g., Fuller et al., 2013; Hays et al., 2003), effectively limiting the predictive value of other extrapolative studies. Are the Current Measures of Primary Sex Ratios Appropriate? There is a lack of empirical measures of primary sex ratios from natural sea turtle nests or rookeries. Primary sex ratios from other turtles (freshwater species such as Chrysemys picta, Trachemys scripta, Chelydra serpentina) are limited to a few multi-year J. Exp. Zool. (Mol. Dev. Evol.)

300 estimates that could serve for comparisons (Janzen, '94; Schwanz et al., 2010). For marine turtles, verified sex ratios from the field were usually from one region (Georgia, USA) or sampled over few seasons or parts of seasons (Mrosovsky and Provancha, '89, '92; LeBlanc et al., 2012). That data deficiency represents a significant challenge for understanding relationships between environmental (temperature) perturbation and sex ratio outcome. It also limits the applicability of sex ratio estimates for the management of imperiled species. This situation prevailed because (until recently) nonlethal sex identification methods were unavailable, logistically inappropriate for natural nests (e.g., Gross et al., '95), or expensive (Wyneken et al., 2007). Regulatory limitations that typically set how many turtles may be sampled, sampling frequency, and logistics often prevent sampling for the entire season or multiple years (Limpus et al., '85; Godfrey and Mrosovsky, '99; Wibbels, 2003; Wyneken et al., 2007; Mrosovsky et al., 2009; Santidri
an-Tomillo et al., 2014). Consequently, samples are often incomplete, not representative of a season's sex ratios and do not account for “atypical” years (Godfrey and Mrosovsky, '99). For long-lived species like marine turtles that reproduce multiple times annually across a long reproductive life, the hatchling sex ratio of an “atypical” year can be a less important component of a female's reproductive effort than the sex ratios of all the hatchlings that she produces over her lifetime. Cumulative primary sex ratios are likely the outcome of each female's iteroparous reproductive effort. Such perspective effectively may be lost to scientists that use a single year's sex ratios as the basis for climatic effect extrapolations. The perspective of what is typical is a necessary part of what makes extrapolation valuable. Are TSD Turtle Species at Greater Risk? The features of climate change identified as relevant threats to marine turtles are nest warming beyond thermal limits of development (Howard et al., 2014; Saba et al., 2012), “feminization” of developing embryos (Hawkes et al., 2007; Fuentes et al., 2009, Escobedo-Galv
an and Gonz
alez-Salazar, 2012; Woolgar et al., 2013), sea level rise, and an increase in the severity of storms that will impact nesting beaches leading to nest and embryo mortality (Pike and Stiner, 2007; Brierley and Kingsford, 2009; Pintus et al., 2009, Fuentes et al., 2010, 2011a). Regional warming is presumed to affect embryos by skewing sex ratios too severely for effective future reproduction, or by heating nests beyond the thermal limits for embryonic survival (e.g., Mrosovsky,’84; Davenport, ’89; Hays et al., 2003; Hamann et al., 2007; Hawkes et al., 2009; Wood, 2014). Sea level rise from thermal expansion and melting ice (Hanson et al., 2006; IPCC, 2007) is expected to eliminate nesting beaches (Drinkwater et al., 2010; Witt et al., 2010). Increased storm intensity may cause increased rainfall during incubation that can suffocate eggs, alter nest temperatures, and increase nest loss from washout or tidal inundation. Yet, some suggest that TSD J. Exp. Zool. (Mol. Dev. Evol.)

WYNEKEN AND LOLAVAR species may have survival advantages (Hulin et al., 2009; Kallimanis, 2010; Silber et al., 2011; Marcovaldi et al., 2014) that will allow them to persist and effectively reproduce (see Table 2 and Fig. 1). In spite of more than three decades of TSD research with marine turtles and the recognition that demographic data are inadequate, the overall issue of adequately sampling primary sex ratios at any location remains. The small sample size challenge, in addition to the limitations associated with use of second and third level proxies leave the state of the science as inadequate for establishing baselines against which to compare past or future years. Here we explore the effects of environmental variations associated with climate change upon marine turtle hatchling sex ratios. We focus on the loggerhead turtle, Caretta caretta, a species with TSD (Mrosovsky and Yntema, '80) as a case study in which we both provide multi-year and across the season sampling, but also invoke the use of some proxies (particularly in relating the temperatures during the TSP to sex ratios of our samples). The loggerhead is a good test subject because it has a semitropical to temperate nesting distribution that is vulnerable to sea level rise risks as well as increasing temperatures. Loggerhead primary sex ratios are important in Florida (USA) because about 85% (>4 million hatchlings/year) of North Atlantic Loggerhead turtles (a Distinct Population Segment [DPS]) hatch in Florida (Conant et al., 2009, TEWG, 2009, USFWS and NMFS, 2011). Florida's coastline is designated as a critical habitat for nesting loggerheads (USFWS and NOAA, 2014) and those beaches are very high risk from projected sea level rise (Fig. 2). We sequentially discuss the field studies then experimental laboratory studies. Methods of Sampling Nest Temperatures and Primary Sex Ratios in the Field We systematically sampled loggerhead primary sex ratios across 10 years to test for annual consistency and assess nest incubation temperatures empirically in the field. We necessarily used several proxies in our study because several empirical measures were either not available, limited, or not permitted. Consequently, we compared loggerhead sex ratio samples to laboratory-derived temperature-sex ratio functions (particularly Mrosovsky, '88 nest G data and the species’ summary by Wibbels, 2003; Fig. 3A). Because of logistical and regulatory limitations on nest sample sizes, we chose to sample the rookery longitudinally rather than characterize nest sex ratios. Nests (N ¼ 11–14) were marked across the entire nesting season. More nests were marked than provided turtles to accommodate for nest loss to predators, storms, equipment failure, and unplanned hatchling releases. The sample sex ratios together were obtained from nests during the first, middle, and last quartiles of the nesting season, with no less than three nests representing each quartile. Sex ratios were averaged by year and the 10-season average was based on the 102

Dermochelys coriacea

Chelonia mydas

Caretta caretta

Species

Lab and field comparison Inferred from nest temperatures and pivotal temperatures on a different beach (Chevalier et al., '99)

Field measures Summarized temperature ranges across season Related sex ratio to incubation temperature and compared methods of estimating hatchling sex ratio. Needed to use empirically derived beachspecific temperature adjustments Lab. Constant temperature incubation Model estimation

Field study-examined nest temp (daily means) Field study-sand temperature

Field measures Field versus Lab examined incubation duration to establish incubation duration/sex ratio curve for field Model-establishes CTE (constant temperature equivalent), used constant temp, cyclic temp, developmental rate Field sex ratios modeled from air temp & sand temp to est. sex ratio. Thermal limits modeled from Ackerman ('97)

No

Model-incubation duration to hatchling sex ratio derived from artificially incubated eggs from a rookery 80 km away based in (Mrosovsky et al., 2002 lab) Field Measures

Yes Yes No

Yes. Histology No

Yes No Yes, 3–9 live hatchlings were sexed

No Yes-microscopic examination of sections of gonads

No

Yes

Yes Yes

Yes

No

Verification at rookery

Model-incubation durations and sand temperatures

Method

Summary

Effects of rain inferred

Empirical validation

Verified different regions of nest. small sample sizes Sex ratios inferred from temperatures One season. Sex ratios borrowed from several other locations to develop relationship

Sex ratios predicted from published pivotal temperatures, from air and sand in different year. Discussion is prudent, notes rainfall, position on beach, etc. Dataloggers in top, middle, bottom of nest 1986-1988 In 1988-sex was not verified due to female bias past two years

Calculated future sex ratios based on air temp rise predictions Model-Air temperature correlates strongly with sand temperature in areas with little rain. Compared summer air temperatures to past years for future estimations Estimated sex ratios from dead in nest and 10 euthanized hatchlings. No match Verified different regions of nest. small sample sizes Sex ratios from incubation durations

Table 2. Literature that reports hatchling sex ratios with and without site-specific verification of those sex ratios.

Citation

(Continued)

Binkley et al. (’98) Santidri
an Tomillo et al. (2014) Mrosovsky ('82) Dutton et al. ('85) Houghton et al. (2006)

Kaska et al. ('98) Standora and Spotila ('85) Godley et al., 2002

Hanson et al. ('98) Mrosovsky and Provancha ('89, '92)

Hawkes et al. (2007)

Kaska et al. ('98) Godfrey and Mrosovsky ('97); Mrosovsky et al. ('99) Georges et al. ('94)

LeBlanc et al. (2012)

Zbinden et al. (2007)

Katselidis et al. (2012)

ENVIRONMENTAL SEX DETERMINATION IN LOGGERHEAD TURTLES 301

J. Exp. Zool. (Mol. Dev. Evol.)

Summarized by Standora and Spotila ('85) Wibbels (2007)

Dead in nest or inferred from nest temperatures

Sex ratios inferred fro temperatures

J. Exp. Zool. (Mol. Dev. Evol.)

Lepidochelys olivacea

No

Some

Summarized temperature ranges across season

Reviewed lab and field. Few natural nest sex ratios

Yes

Dead in nest or inferred from nest temperatures. Most address hatchery sex ratios without natural nest sex ratios for comparison

Wibbels (2007)

Sex ratios inferred from temperatures No Lepidochelys kempii

Strong female bias

Summarized various experimental and natural temperature ranges across season Artificially incubated eggs used to define curve to apply to field nests. Field relocated nests Reviewed lab and field Few natural nest sex ratios

Some

Summarized by Standora and Spotila ('85) LeBlanc et al. (2012)

1 season 49 of 51 nests

Used dead in nest from incubators

Wibbels et al. ('99)

27 years, multiple beaches

Yes Initially yes then used curve as model Used dead in nest Lab curve, field application Incubation duration applied from one verified beach to incubation duration at other beaches Eretmochelys imbricata

Species

Table 2. (Continued)

Method

Verification at rookery

Summary

Citation

WYNEKEN AND LOLAVAR

Godfrey et al. ('99) Marcovaldi et al. (2014)

302

Figure 1. Predicted effects of climate change on marine turtle population survival.

sex ratios. The sex of each turtle in our samples was verified morphologically using highly accurate laparoscopic techniques (Wyneken et al., 2007). The rookery was defined by the Northern and Southern county lines of Palm Beach County (Juno Beach and Boca Raton) located on the east coast of peninsular Florida, U.S.A. The same tagged turtles have been observed using both locations for nesting (Wyneken, unpublished data) so the two beaches were treated as a single site. Incubation temperatures were measured using temperature loggers (HOBO model H8; resolution 0.4°C, accuracy  0.8°C, 2002–2009 and HOBO model U22-001; resolution 0.02°C, accuracy  0.21°C, 2009–2013) placed in the center of each clutch. The loggers were calibrated prior to use. The temperature loggers were programmed to take readings every 15 min (U22001) or every hour (H8) throughout incubation. Temperature loggers were placed in the egg chamber either during egg deposition after approximately 50 eggs were laid, or we opened nests (prior to 0800 hr) that were deposited the previous evening, removed the top three egg layers (
50 eggs), inserted the temperature logger in the middle of the egg mass, replaced the eggs in the order they were removed, and resealed the nest with the same sand that covered the eggs initially. We recovered the temperature loggers during post-emergence nest inventories. Temperature data were used from the middle 1/3 of the incubation duration (a proxy for the TSP); total incubation duration was defined as the morning the nest was marked to the day the first hatchlings emerged. The mean, minimum and maximum temperatures were identified and used to characterize TSP temperatures.

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Figure 2. Areas of Florida at risk of inundation due to rising seas. Most of the major sea turtle rookeries are in areas that are 2–5 m above sea level, however nesting turtles use the seaward beach face and so nests are particularly vulnerable. White lines denote areas with large nesting concentrations. Thicker lines are associated with greater nesting densities and are based on relative abundance of clutches as summarized by SWOT (http:// seamap.env.duke.edu/swot, accessed 13/07/2014; Kot et al., 2014). Sea level image created by Robert A. Rohde / Global Warming Art (with permission).

Hatchlings (n ¼ 7–10 turtles/nest) were collected from nests during or a few hours just prior to an emergence using standard methods (Salmon et al., 2012; Wyneken and Salmon, '92). We collected only physically normal “first emergence” hatchlings from each nest as a sample to provide each sex ratio. Turtles were reared at the Florida Atlantic University Marine Laboratory to a mass of 120 g, a size that enabled us to identify their sex laparoscopically (Wyneken at al., 2007). The turtles were retained at the lab for 1 week, post-laparoscopy, to ensure recovery and then were released offshore in the Florida Current (see Mansfield et al., 2012, 2014).

Figure 3. Comparisons of lab-derived loggerhead sex ratios and field-derived sex ratios from natural nest samples. A: Sex ratios from two clutches (F and G) of Caretta caretta eggs incubated at constant temperatures in the lab show that there are clutchspecific differences in sex ratio response that had unknown causes (from Mrosovsky, '88). B: Generalized model of cooler M warmer F sex determination in sea turtles from Mrosovsky and Godfrey (2010). For loggerheads in Florida, the pivotal temperature is 29°C, based on laboratory studies. C: Observed sample sex ratios from Palm Beach County Florida. Note that samples that incubated in situ do not fit the typical sigmoid relationship.

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Figure 4. Mean sample sex ratios (bars), averaged across all samples each year. A: There is a strong female bias in most years. When temperatures during turtle nesting season were above normal (red), the years are designated as “Hot.” When rainfall was above average for the same periods, bars are blue and years are designated as “Wet.” Green designates a normal year. The red and blue bar was a hot and exceptionally wet year. N ¼ number of samples each year. Note that just one normal year occurred during the 10 years of this study. B: Sample sex ratios by year differ among years. Most samples that produced males were collected in wet years. In 2004, the last three nest samples were lost to hurricanes; in 2011 the last six nest samples were lost to tropical storm surge so the sex ratios represent the early and middle parts of the season. Numbers in the diamonds are the counts of each sex ratio. In 2002, both Juno Beach and Boca Raton were sampled leading to the large numbers of samples.

Results of Field Nest Temperatures and Sex Ratios The sample sex ratios obtained in field are presented in Figure 4 and Table 3. Nests were fitted with temperature data loggers from April–August to represent the entire nesting season. On several occasions instruments failed to record the full incubation duration and were omitted from this part of the study. During several seasons, particularly 2004, 2011, 2012, tropical storm surge and shore break waves destroyed many natural nests including our instrumented nests. However, we J. Exp. Zool. (Mol. Dev. Evol.)

consider the resulting sample sex ratios across all seasons (n ¼ 656 turtles) as representative for the study site because instrumented nests that survived were near other natural nests that survived. The subset of samples (n ¼ 55) used in this analysis had full thermal data and provided 7–10 hatchlings each. The means of the samples sex ratios were female-biased each year. We found that years with greater rainfall than average tend to produce more male hatchlings, regardless of temperature

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Table 3. Sex ratio samples for Caretta caretta at two beaches Boca Raton and Juno Beach Palm Beach County, Florida USA. Year

Nests sampled

Turtles

Sample sex ratio (% F) mean, range

Mean TSP temperature temperature range of TSP °C

2002

13 BR 11 JU 9 JU 9 BR 8 BR 3 BR 7 BR 13 BR 7 BR 12 BR 10 BR

126 118 86 78 16 24 70 117 92 95 90

87%, 57–100% 7 mixed; 6 all F 91%, 56–100% 6 mixed; 5 all F 95%, 88–100% 4 mixed sex; 5 all F 89%, 11–100% 1 mixed sex, 8 all F 100% 100% 71%, 80–100% 4 mixed sex, 3 all F 100% 100% 74%, 0–100% 2 all M; 1 mixed sex, 9 all F 65%, 0–100% 1 all M; 3 mixed sex; 6 all F

30.6, 26.0–35.3 30.8, 24.4–35.3 32.8, 29.9–35.7 No thermal data No thermal data No thermal data No thermal data 32.3, 29.1–35.2 32.4, 29.7–36.5 30.8, 24.5–34.2 30.6, 26.8–35.9

2003 2004 2007 2008 2009 2010 2011 2012 2013

In 2002, both Boca Raton (BR) and Juno Beach (JU) were sampled and their sex ratios and nest thermal environments were statistically indistinguishable. Juno Beach was sampled alone in 2003 and Boca Raton was examined alone in 2003, and 2007–2013. Mean temperature during the estimated TSP is the grand mean of the samples by year. The ranges are the min and maximum incubation temperatures during the estimated TSP for samples at the site each year. In 2004, 2007 and 2008, temperature data loggers were not placed in loggerheads nests that provided the samples so sex ratios are presented without associated incubation temperatures during the presumed TSP.

within and above the literature pivotal temperature and TRT for the species (see Wibbels, 2003 for review; Blair 2005). In four of the years (2007, 2008, 2010, 2011), all the samples yielded 100% females. During 2010 and 2011, mean nest temperatures during the TSP were high. Six seasons (2002–2004, 2009, 2012, 2013) produced mixed sex (both male and female turtles) samples. All male or mixed sex samples were more common in wet years. In 2002, June and early July received much greater rainfall levels than normal. There were 13 mixed sex samples of 24 collected that were incubating during this period. One year (2003) was warmer than normal but was also wetter than normal (NWS, 2003). The mean sex ratio of that year's samples was femalebiased however, four of nine samples produced both male and female hatchlings. In 2009, the nesting season (May–July) was one of the wettest (NWS, 2009); four of seven samples were mixed sex. In 2012, near record high rainfall occurred and air temperatures were slightly below normal due to cloud cover and rain (NWS, 2012). During the 2013 rainy season, rainfall was significantly above normal through the first half of the nesting season and near normal for the second half; it was the 2nd warmest year on record (NWS, 2013). We found 100% male samples in 2012 (a wet year, 2 samples) and 2013 (a wet hot year, 1 sample). These were the only all male samples found across the 10 years and 102 samples and so they set the 2012 and 2013 seasons apart from all others. Table 3 summarizes incubation temperatures during the middle third of incubation. Very few years produced samples incubating within the lower range of the TRT (2002, 2012, 2013); all were years with higher than normal rainfall. In hot years, the

incubation temperatures exceeded the published pivotal temperature for C. caretta (Wibbels, 2003). The 2003 nesting season was hotter than normal and both mean TSP incubation temperature and the TSP range (Table 3) were well above the pivotal temperature for Florida loggerheads (29°C) yet four of nine samples were mixed sex. Conclusions. When sex ratios were plotted as a function of mean estimated TSP temperature, the results suggest that the sample sex ratio responses in the field do not fit the logistic sex ratio response function that Godfrey and Mrosovsky ('99) derived from lab studies for the same species. The distribution of 100% female nests spans more than 6°C and does not resemble the laboratory responses (Fig. 3A) or the predictive model (Fig. 3B). When logistic regression models were fit, the distributions of sex ratios versus mean incubation temperatures (middle third of incubation) and the resulting curves were not significant predictors of the sex ratio (r2 ¼ 0.27). This is a poor fit related to multiple sex ratios at any one temperature, the same sex ratios occurring at multiple mean temperatures, and the lack of any sex ratios anchoring the curve below 28°C (Fig. 3C). Our field data suggested that incubation temperature, alone, failed to explain the nest sex ratios. Further, the apparent association of male production in years with heavy rainfall led us to explore the possibility that sex ratios were determined by an interaction between temperature and nest moisture. To test for such an interaction, we designed laboratory experiments where we had greater control over both nest temperature and nest moisture content during the entire incubation period. J. Exp. Zool. (Mol. Dev. Evol.)

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Methods of Testing the Experimental Effects of Nest Moisture on Sex Ratios We collected 30 eggs from each of 10 loggerhead nests in Boca Raton, Florida the morning following their deposition. We transported them in a climate-controlled vehicle to the lab less than 30 min away. The eggs from each nest were divided into three groups of 10 eggs each. The eggs were placed in StyrofoamTM “nest boxes,” in two layers of five eggs that were in contact with one another. They incubated in sterilized local sand from the same part of the beach used for nesting. Sand thickness was
3.5 cm at the bottom, 4–6 cm on the sides, and 3.5 cm on top. Each nest box (20  15  23 cm3 internal dimensions) was assigned to one of three incubators set at 29.4°, 30.4° or 31.4°C ( 0.2°C). Each incubator held 10 nest boxes (100 eggs total/incubator). All of the nest boxes were positioned at the same level in the incubator and exposed to high moisture levels. Incubator temperature and sand temperature at egg level were measured using HOBO U22-001 temperature loggers. Egg temperature was not measured directly. Each incubator was equipped with a 150 W incandescent lamp as a heat source, a fan-assisted evaporative moisture system (Walgreens Cool Moisture Humidifier Model 890-WGN), and two 15 cm fans that circulated the heat and moisture so that the conditions were uniform for all nest boxes throughout the chamber. The heat and moisture system were controlled (Omega. 1 com iSeries Temperature & Process Controller Model CNi32, Stamford, Connecticut). Temperature and humidity of each incubator were monitored throughout the study using HOBO U12

Temperature & Relative Humidity data loggers ([accuracy  0.35° C and resolution 0.03°C from 20 to 40°C] Onset Corp., Cape Cod, Massachusetts). Nest boxes were watered as needed by spraying the surface with a Di-H2O mist when sand moisture levels dropped below threshold levels; the water was allowed to percolate through the sand. The volumetric sand moisture (m3/m3) in each nest box was measured with Decagon EC-5 soil moisture probes fitted to HOBO Micro Station Data Loggers (Onset Computer Corp. Model H21002, resolution 0.0007 m3/m3 (0.07%), accuracy  0.031 m3/m3 ( 3.1%) per manufacturer's specifications). Sand moisture ranged across 0.05–0.12 m3/m3 over all of incubation corresponding to moist beach sand associated with loggerhead turtle nests (Table 4). The threshold for adding water to the sand was 0.05 m3/m3. On rare occasions sand moisture levels in single nest boxes dropped lower for less than a day. Sand moisture levels increased prior to hatching as eggs released moisture. All live hatchlings were brought to the lab and reared to 120 g then sexed laparoscopically. The procedures were the same as for natural nest samples. One dead late-term embryo was sexed histologically. Sex ratios were calculated by nest box and by incubator condition. The nest box sex ratios were compared with expected sex ratios calculated from two different estimators: (i) The laboratory temperature/sex ratio responses curve for Floridian loggerhead turtle eggs that were collected approximately 75 km north of our Boca Raton study site (Mrosovsky, '88) and (ii) expected sex ratios based upon incubation duration (after Mrosovsky et al., '99).

Table 4. Loggerhead sex ratios (percent female [F]) from high moisture conditions in the laboratory. 29.4°C Incubator N

Obs %F

Expect %F

10 8 10 8 10 9 10 10 9 Mean

0 0 0 50 0 0 0 60 0 12%

60 60 60 60 60 60 60 60 60

30.4°C Incubator

Kruskal–Wallis

N

Hadj ¼ 13.1 1 df P < 0.0003

10 9 10 10 8 10 10 9 8

Obs %F

Expect %F

0 0 70 0 0 30 0 0 0 11%

75 75 75 75 75 75 75 75 75

31.4°C Incubator

Kruskal–Wallis

N

Hadj ¼ 15.6 1 df P < 0.0001

10 8 10 10 10 10 8 10 10

Obs %F

Expect %F

100 88 100 100 100 100 100 100 100 99%

90 90 90 90 90 90 90 90 90

Kruskal–Wallis Hadj ¼ 9.8 1 df P < 0.002

Expected percentage female, based on two loggerhead temperature-sex ratio response curves in the literature (Mrosovsky, '88; Yntema and Mrosovsky, '79), were 29.4°C: 60%, 30.4°C: 75%, 31.4°C: 90%. The observed % F results were not normally distributed and the expected values lack a measure of variability so Kruskal–Wallis comparisons were used to analyze the results. The observed sex ratios differed from expected in all three treatments.

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Results From Laboratory Experiments of High Nest Moisture and Nest Temperature All incubators maintained the set temperature  0.2°C except when new nest boxes were positioned, then air temperatures dipped briefly (less than 3 min) 0.6–1.0°C. All eggs were collected over an 11 day period. In the morning and within a few hours of deposition, the eggs were placed in nest boxes, and then in their incubators; all nest boxes were at the same level. Relative humidity either stayed the same or increased a few percent to 100% as the investigator perspired). Hatching success was high (94% overall). Eggs of one female failed to develop in any of the incubators; the remaining eggs from her clutch that were left in the natural nest, also failed to develop. Consequently, each incubator produced nine sex ratios, (one for each nest box). There were strongly male-biased sex ratios from the 29.4°C and 30.4°C incubators and strongly female-biased sex ratios at 31.4°C. Loggerhead sex ratios in each incubator differed from predicted sex ratios (Fig. 5, Table 4). Seven nests incubating at 29.4°C produced 100% male hatchlings incubating in 58–61 days. These sex ratios are roughly consistent with the laboratory-derived response function but not that derived from the field (Fig. 5). Two nests at 29.4°C produced mixed sex samples (50% F at 60 days

307 and 60% F at 62 days). The 60 day nest is close to the field-derived expected response curve. There was another nest box in the same incubator that also incubated in 60 days but produced no females. The 60% sex ratio falls outside of either sex ratio incubation duration response curve. Seven nests incubating at 30.4°C produced 100% male hatchlings that completed incubation in 52–58 days. These male turtles incubated over much shorter incubation durations than predicted from either the laboratory or field derived sex ratio response curves. Two nests at 30.4°C produced mixed sex samples (30% F at 52 days and 70% F at 54 days). The 54 day sex ratio appears to be an outlier for that set of experimental conditions, but it actually is consistent with the prediction generated by the laboratory-derived response curve. The 52 day sex ratio falls well below what is predicted by either curve. Eight nests incubating at 31.4°C produced 100% female sex ratios across 47–52 days. These results are consistent with the field derived incubation duration predictions. The ninth nest produced 88% female at 49 days and so departed from both the field and lab predictions by producing a single male. Conclusions. Together these experimental results support what we hypothesized based upon our field data, specifically, that temperature alone does not accurately predict turtle sex ratios. The effect is especially pronounced when high moisture levels interact with extremely warm incubation temperatures.

DISCUSSION

Figure 5. Sex ratios from laboratory incubated 10-egg “clutches” that were maintained in sterilized sand at high moisture conditions, presented as a function of incubation duration (which is correlated with incubation temperature). Dot sizes represent the numbers (1, 2, 3, or 4) of sex ratios at a given temperature (increasing dot size represents more “clutches” showing the same sex ratio). The predicted sex ratio versus incubation duration function is from Godfrey and Mrosovsky ('99). Dashed line is the derived field curve; solid line is the derived laboratory curve. Note that the temperatures we used should have produced predominantly female-biased sex ratios but that result occurred only under the warmest (31.4°C) conditions and usually more rapid incubation.

This study critically reviews the literature that suggests turtle species with TSD are particularly vulnerable to the effects of climate change because of two presumed risks: feminization of their clutches, and nesting sites that would become too warm, too wet, or too unstable to support incubation. We approached our analysis by asking whether these claims are well supported. That question led us to assess the accuracy of the various proxies used to estimate sex ratios. We conclude that in many cases, the proxies when applied to sites we sampled, would have yielded sex ratio estimates that bore little to no resemblance to the actual sex ratios expressed in the nests because the estimators did not account for developmental variability, variable temperatures, and the effects of temperature and moisture together. Under those circumstances, it became obvious that scientists are not yet in a position to predict how climate change will affect the primary sex ratios due to a lack of data or unsupported correlations. The importance of accurately determining hatchling primary sex ratios lies in their value for identifying changes decades before their reproductive consequences are realized. Yet, the ways primary sex ratios are estimated have moderate to extreme limitations because many are based on weak correlative and predictive model assumptions. While all primary sex ratios are estimates and most are based on proxies, we can identify how the proxies differ in quality by how many steps are inferred between the presumed key environmental variable(s) and the incubation J. Exp. Zool. (Mol. Dev. Evol.)

308 environment experienced by the embryo. Girondot et al. (2010) discusses how this concept is applied to reptile sex ratio issues in detail, and importantly reviews how few proxies are tested quantitatively and how they typically ignore embryonic growth and physiology. We address three common limitations in our analyses with (i) sample sex ratios collected across many nesting seasons, (ii) samples collected across the entire nesting season, and (iii) verification of all the turtle sexes that are included in the analyses. We presented the results of our empirically sampled sex ratios across 10 nesting seasons to provide a baseline that includes normal, hot, wet, and dry weather. The sex ratios (N ¼ 102 samples, 3–13 samples/site/year, from 656 turtles) are the consequences of the incubation environments in natural nests. They were sampled for a key loggerhead rookery in Palm Beach County, Florida, which is part of the Peninsular Florida Recovery Unit (Conant et al., 2009). Our sample sex ratios from the field provide an empirically based estimate of primary sex ratios at this rookery over time. Sex ratios were moderately to highly female-biased in all years. Our results are consistent in this characteristic with previous studies of Florida's loggerhead nests and nesting beaches based on thermal proxies, hatchling sampling over parts of the season, dead-in-nest hatchlings, or laparoscopically verified small samples (Mrosovsky and Provancha, '89; Mrosovsky and Provancha, '92; Mrosovsky et al., '95; Hanson et al., '98; Blair, 2005; Rogers, 2013). Year-to-year differences in sex ratios were large sometimes (e.g., 2009 differed from 2010 by 29%, Table 3), and ranged from 65 to 100% female. Within seasons that produced both male and female hatchlings, the sample sex ratios varied from 0 to 100% female. Five of the 6 years that produced male hatchlings were years with heavy rainfall during at least the first half of incubation. This important observation leads us to hypothesize that the effects of temperature on sex determination can be modified by moisture. Various studies have identified phenotypic effects of moisture on several aspects of embryonic growth, embryo physiology, hatchling size, and organ growth in turtles (e.g., Morris et al., '83; Packard et al., '87; McGehee, '90; Janzen, '93). Just two have documented impacts of moisture (rainfall [Godfrey et al., '96] or ocean overwash [Foley et al., 2000]) on sex ratios in the field. Our distribution of sample sex ratios versus mean incubation temperature did not resemble the logistic (sigmoidal) relationship found in constant temperature incubation studies (Mrosovsky, '88). At the 29°C pivotal temperature for loggerheads (Mrosovsky, '88), we found a number of sex ratios, most of which differed greatly from 1:1. At temperatures that would be predicted to produce 100% female sex ratios, we found mixed sex samples. Several studies note that mean TSP temperatures explain only a portion of the variation sex ratios in natural nests. Girondot et al. (2010) point out that the mean TSP incubation temperature and mean pivotal temperatures do not account for variation in embryonic growth rates or the physiology of sex determination J. Exp. Zool. (Mol. Dev. Evol.)

WYNEKEN AND LOLAVAR so mean temperatures also can be considered a proxy. Georges et al. (2004) explain that while both mean temperature and temperature fluctuations influence developmental rates, as temperatures fluctuate it becomes more difficult to discern what threshold temperatures determine a male or female outcome. However, in the case of sea turtles, variation in environmental temperatures at nest depth tends to be very low (