Advertisement
Browse Subject Areas
?

Click through the PLOS taxonomy to find articles in your field.

For more information about PLOS Subject Areas, click here.

  • Loading metrics

Female-Bias in a Long-Term Study of a Species with Temperature-Dependent Sex Determination: Monitoring Sex Ratios for Climate Change Research

  • Joanne Braun McNeill ,

    joanne.b.mcneill@noaa.gov

    Affiliation Southeast Fisheries Science Center, National Marine Fisheries Service, National Oceanic and Atmospheric Administration, Beaufort, North Carolina, United States of America

  • Larisa Avens,

    Affiliation Southeast Fisheries Science Center, National Marine Fisheries Service, National Oceanic and Atmospheric Administration, Beaufort, North Carolina, United States of America

  • April Goodman Hall,

    Affiliation Southeast Fisheries Science Center, National Marine Fisheries Service, National Oceanic and Atmospheric Administration, Beaufort, North Carolina, United States of America

  • Lisa R. Goshe,

    Affiliation Southeast Fisheries Science Center, National Marine Fisheries Service, National Oceanic and Atmospheric Administration, Beaufort, North Carolina, United States of America

  • Craig A. Harms,

    Affiliation Center for Marine Sciences and Technology, College of Veterinary Medicine, North Carolina State University, Morehead City, North Carolina, United States of America

  • David W. Owens

    Affiliation University of Charleston, South Carolina at the College of Charleston, Charleston, South Carolina, United States of America

Female-Bias in a Long-Term Study of a Species with Temperature-Dependent Sex Determination: Monitoring Sex Ratios for Climate Change Research

  • Joanne Braun McNeill, 
  • Larisa Avens, 
  • April Goodman Hall, 
  • Lisa R. Goshe, 
  • Craig A. Harms, 
  • David W. Owens
PLOS
x

Abstract

Alterations have occurred and continue to manifest in the Earth’s biota as a result of climate change. Animals exhibiting temperature dependent sex determination (TSD), including sea turtles, are perhaps most vulnerable to a warming of the Earth as highly skewed sex ratios can result, potentially leading to population extinction resulting from decreased male recruitment. Recent studies have begun to quantify climate change impacts to sea turtle populations, especially in terms of predicting effects on hatchling sex ratios. However, given the inherent difficulty in studying sex ratios at this life stage, a more accurate assessment of changes in population sex ratios might be derived by evaluating the juvenile portion of foraging aggregations. We investigated the long-term trend in sex ratio of a juvenile loggerhead (Caretta caretta) sea turtle population inhabiting Pamlico and Core Sounds, North Carolina, USA. We used plasma testosterone reference ranges measured using radioimmunoassay (RIA) to assign sex for 959 turtles and confirmed sex assignment of a subset (N = 58) of the sampled turtles through laparoscopic examination of their gonads. Our results demonstrate that for this particular population of loggerheads, sex ratios (3Females:1Male) had not significantly changed over a 10 year period (1998–2007), nor showed any significant difference among 5-cm straight carapace length (SCL) size classes. Ultimately, these findings provide a basis for comparison with future sex ratios, and highlight the importance of establishing similar long-term studies monitoring secondary, rather than primary, sex ratios, so that needed mitigation measures to climate change impacts can be implemented.

Introduction

Since the mid-nineteenth century, a notable increase (0.6–0.8°C) in the Earth’s mean surface temperature has been documented and is continuing at an accelerated rate [16]. As a result of this warming trend, changes have occurred and continue to manifest in the Earth’s biota including phenology (timing of seasonal activities) and physiology of organisms, range and distribution of species, composition of and interactions within and among communities, and structure and dynamics of ecosystems (see [4, 6] for a review). Animal and plant phenology and geographic distribution are processes most commonly monitored to assess population response to climate change. In a review of the studied phenologies of 1,598 species, it was estimated that more than half (59%) had demonstrated noticeable changes during the past 20 to 140 years [7]; earlier breeding (or first singing) of birds, flowering of plants, and spawning of amphibians are among the most common changes observed for plants and animals [7]. However, in some cases, more extreme changes can occur with these temperature increases, such as invasions of warm-water species and the eradication of other species, which can have a greater impact on populations [4]. For example, in the tropics, increases in water temperature of less than 1°C can result in large-scale coral bleaching and ensuing mortality of all coral species [4].

Likewise, for those animals more susceptible to alterations in the thermal environment, a change in climate might have more adverse effects on a population [4]. In the case of ectotherms, whose survival and reproduction are strongly dependent upon thermal conditions, climate change could have serious consequences. For many reptile species, the temperature at which the eggs are incubated determines the sex of the hatchlings, a process known as temperature-dependent sex determination (TSD) [reviewed in [8]]. During a 5-yr study monitoring air temperature and sex ratios of hatchling painted turtles (Chrysemys picta), empirical evidence of the sensitivity of a species with TSD to even slight changes in temperature was provided [9]. The direct relationship between annual hatchling sex ratios and mean July air temperature was documented, which in some years produced 100% male or female hatchlings. Similar to painted turtles, sea turtle female offspring are produced at higher temperatures with male offspring produced at lower temperatures [10]. Thus, if the predicted rise in global temperatures results in a disproportionate number of female hatchlings and similarly skewed adult sex ratios, single-sex populations could be produced, eventually leading to demographic collapse [9, 11, 12].

While many researchers have recognized the potential negative effect climate change could have on sea turtle populations [13], only recently have studies begun to quantify these impacts, especially in terms of predicting skewed hatchling sex ratios [1418]. Consequently, long-term monitoring of nesting sites was recommended so that potential future changes to primary (hatchling) sex ratios could be measured [19, 20] and necessary mitigation measures implemented in a timely manner. However, primary sex ratios are influenced by a variety of factors, including geographic location of nests, time of year eggs are laid, substrate in which eggs are laid, metabolic heating, and annual weather patterns [2126], and this variation can occur within the same beach over several years [21, 27]. Moreover, primary sex ratios are generally estimated from the sex of a limited number of dead or live hatchlings salvaged from a few nests, or inferred from measured incubation temperatures or durations [26], which requires obtaining pivotal temperatures in a laboratory setting, as well as sacrificing a sufficient number of hatchlings for validation [20]. While non-lethal methods to sex post-hatchling turtles have been developed, they are expensive and logistically difficult [28].

Alternatively, assessing the sex ratios of neritic juveniles (secondary sex ratio) might offer a better way of tracking trends in a population’s sex ratio [21, 29, 30]. Since the juvenile life stage in sea turtles is protracted, it represents many cohorts and, consequently, many years of hatchling production, incorporating sex ratio variability over time [30, 31]. Moreover, random sampling of populations is possible, as neritic juveniles are less likely to have sex-specific behavioral biases displayed by adults [21, 30]. Finally, methods used to sex juveniles are direct and non-lethal—gonads of stranded dead turtles are examined during necropsy or testosterone levels in the blood of live turtles are measured. When combined with laparoscopic verification of RIA results, blood testosterone can be an accurate means of determining sex of large numbers of juveniles [32]. Despite logistical difficulties in accessing neritic juveniles, estimated sex ratios at this stage might reveal a more accurate representation of a sea turtle population’s future reproductive potential and response to changes in the environment [21, 32].

As part of an ongoing assessment of sea turtle populations along the US Atlantic coast [33], we investigated the long-term sex ratio of neritic juvenile loggerhead (Caretta caretta) sea turtles inhabiting Pamlico and Core Sounds, North Carolina, USA. We measured circulating testosterone levels with radioimmunoassay (RIA) and verified sex of a subset of the sampled turtles through visual examination of their gonads to establish a range of testosterone concentrations associated with each sex. We then compared estimated sex ratios over a 10-year period (1998–2007) and among 5-cm straight carapace length (SCL) size classes to determine if significant changes in sex ratios were occurring, potentially due to changes in the climate.

Materials and Methods

This research was conducted under the authority of U.S. Endangered Species Act Section 10(a)(1)(A) scientific research permits from the U.S. Fish and Wildlife Service (#TE-676379) and the National Marine Fisheries Service (#1260) which reviewed and approved all handling protocols for the animals in this study, including blood sampling.

Study area and capture technique

We sampled loggerhead turtles captured in commercial fishing gear (pound nets and long haul seines) in Core and Pamlico Sounds, North Carolina, USA (Fig 1) from 1998–2002 [34] and from 2003–2007 (this study). Pound nets are a stationary gear that passively capture targeted fish by directing them into an enclosure (pound) by means of the lead [35]. Long-haul seines are 1 km x 2 m nets pulled between two boats for up to 8 km before the catch is encircled and concentrated by pulling the net around a stake [36]. When sea turtles are also incidentally captured, they are accessible for sampling purposes. We analyzed turtles sampled only during the summer months (June–August) to increase the likelihood of sampling the resident population and to avoid any seasonal bias in testosterone concentration [34]. Using a sterile syringe with a 3.81 cm, 20 gauge needle, we collected 5 ml of blood from the dorsocervical sinus of the turtle [37]. Blood samples were immediately transferred to a sterile lithium heparin or sodium heparin tube and stored on ice for a maximum of 5 h (i.e., for the rest of the field day). We applied Inconel metal alloy size 681 self-piercing tags (National Brand and Tag Company, Newport, Kentucky, USA) to both rear flippers and injected Passive Integrated Transponder (PIT) tags (Destron-Fearing Corp., South St. Paul, Minnesota, USA, 125 kHz) in the triceps superficialis muscle of the left front flipper to recognize recaptures. We measured standard straight-line carapace length (SCL) from the nuchal notch to the posterior tip to the nearest 0.1 cm using calipers, and excluded turtles with a carapace length greater than 76 cm [30] from the analysis to prevent use of adults in this study (and avoid bias associated with various sex-specific behaviors of the adult population) [30, 31]. We recorded surface water temperature to the nearest 0.5°C using calibrated thermometers. If we encountered previously sampled turtles (from prior years), we included them, as they also were members of that year’s resident population. After returning from the field, we centrifuged blood samples for 6–10 min, then pipetted 2 ml samples of plasma into cryogenic vials, and stored them in an ultra-cold (–80°C) freezer.

thumbnail
Fig 1. Map of study area where juvenile loggerhead (Caretta caretta) sea turtles were captured in pound nets and long haul seines fished in Core and Pamlico Sounds, North Carolina, USA, June to August, 1998–2007.

https://doi.org/10.1371/journal.pone.0160911.g001

Sexing Technique

We utilized a plasma androgen sexing technique to classify sex of the turtles using a testosterone RIA procedure that was previously validated for loggerhead sea turtle plasma [30, 38, 39]. We confirmed sex for a sub-sample of turtles (N = 58) via laparoscopic examination August 2000 and July 2001 [34] and June 2004 to validate sex classification based on the RIA procedure [32]. We characterized sex by examining the shape and surface of the gonads: ovaries have an irregular shape with an undulating edge and granular surface; testes are elongated in shape with a smooth edge and surface [40]. After verifying sex, we subsequently used testosterone levels of known sex turtles to estimate plasma testosterone ranges for each sex.

Statistical Analysis

We combined previous data (testosterone levels and laparoscopy results) collected 1998–2002 [34] with data collected 2003–2007. To determine if there was a significant difference in sex ratio (% females) among years and 5-cm size classes (SCL) ranging from 40.0–75.9 cm, we used a contingency table analysis [41]. In addition, we computed binomial confidence intervals on the proportion of females for each year and size class.

Results

We sampled blood from 959 loggerhead turtles with SCL ranging from 42.8–75.9 cm and conducted laparoscopies on 58 of them. Of the 946 whose sex was determined, 710 were female and 236 were male (Table 1). Eight turtles did not have carapace measurements, so were not included in the % females across size class analysis (Table 2). Water temperature at which turtles were sampled was 20°C or greater. Plasma testosterone concentrations for all turtles ranged from 0.1–11,420 pg/ml (N = 959). The sex ratio of laparoscoped turtles was 2.9F:1.0M. Plasma testosterone concentrations for laparoscoped females ranged from 6.7–432.0 pg/m (N = 43) while males ranged from 372.0–1884.0 pg/ml (N = 15). There was some overlap in testosterone concentration of males with females: one male (372.0 pg/ml) had a lower testosterone concentration than two of the females (394.1 and 432.3 pg/ml). Therefore, we determined that turtles having testosterone values of 371 pg/ml or less would be categorized female, turtles having testosterone values of 433 pg/ml or greater would be categorized male, and turtles having testosterone values 372–432 pg/ml would be categorized unknown (Fig 2).

thumbnail
Fig 2. Frequency of plasma testosterone concentration of juvenile loggerhead (Caretta caretta) sea turtles captured in Core and Pamlico Sounds, North Carolina, USA, June to August, 1998–2007.

A. that were laparoscopically examined (N = 58) (water temperatures 25–29°C) and B. that were not laparoscopically examined (N = 901) (water temperatures 20–32°C). Vertical lines indicate maximum testosterone titer of 371 pg/ml for females and minimum testosterone titer of 433 pg/ml for males. Sex could not be determined for turtles with values between vertical lines.).

https://doi.org/10.1371/journal.pone.0160911.g002

thumbnail
Table 1. Number of females, males, percentage females (95% confidence intervals) and χ2 values for juvenile loggerhead (Caretta caretta) sea turtles.

https://doi.org/10.1371/journal.pone.0160911.t001

thumbnail
Table 2. Number of females, males, percentage females (95% confidence intervals) and χ2 values across size classes (straight carapace length) for juvenile loggerheads (Caretta caretta).

https://doi.org/10.1371/journal.pone.0160911.t002

We used these testosterone ranges to assign sex to turtles not laparoscoped (N = 901), resulting in 668 females, 220 males and 13 unknowns with a sex ratio of 3.0F:1.0M (Fig 2B). We did not find a significant change in sex ratios (% females) among years (χ2 = 8.3, df = 9, P >0.5; Table 1, Fig 3) or size classes (χ2 = 6.3, df = 7, P > 0.5; Table 2).

thumbnail
Fig 3. Estimated annual percentage male and female juvenile loggerhead (Caretta caretta) sea turtles captured in Core and Pamlico Sounds, North Carolina, USA, June to August, 1998–2007.)

https://doi.org/10.1371/journal.pone.0160911.g003

Discussion

Despite concerns about potential changes in sex ratios of animals displaying TSD because of predicted warming conditions, during this study we found that the sex ratio of juvenile loggerhead sea turtles inhabiting North Carolina inshore waters did not change significantly from 1998–2007, nor was there a significant change in sex ratios among the different 5-cm SCL size classes (Table 2). Studies of juvenile loggerheads captured offshore south of Madeira Island (2000–2006) [42] or in the Mediterranean (2000–2011) [43] likewise did not find a significant difference among annual population sex ratios. However, a decreasing proportion of females in Madeira waters for turtles larger than the 45.0 cm SCL size class was noted [42]. Because this is the size at which turtles start leaving the pelagic habitat for neritic waters, the authors postulated that females are making the shift to neritic feeding grounds before males [42]. Although these long-term sex ratio studies do not reveal any trends over time, these data establish a baseline sex ratio for their respective areas against which results of future sex ratio analyses might be compared.

There are a number of reasons why our study’s population sex ratio may not have exhibited any noticeable trend during this time period. Given the variation in annual hatchling sex ratios [27], along with sea turtles’ long-lived, late-maturing life history, a 10 year period likely is not long enough to reveal any changes occurring in a populations’ sex ratio. Another factor to consider is that loggerheads exhibit delayed maturation, with estimates of up to 45 years [44]; thus, the data yielded by neritic juveniles are representative of what environmental conditions were like approximately 10–20 years ago [45]. Furthermore, despite overall increases in global temperatures [5], long-term mean air temperatures of coastal North Carolina from 1940–2005 actually have decreased [46]. Because of the significant relationship demonstrated between air and sea surface temperature [46], this indicates that hatchlings on these beaches may not have been exposed to warming temperatures during this time period.

In addition to representing environmental conditions from several decades ago, our population of juvenile loggerheads comprises multiple genetic nesting populations that in turn reflect different incubation environments. For instance, a study of the genetic composition of a foraging population in North Carolina, revealed that 80% of the turtles originated from the south Florida nesting population, 12% were from the northeast Florida to North Carolina nesting population, 6% from Yucatan, Mexico, and 2% from other rookeries [47]. While warmer beaches of the south Florida nesting population might be contributing a high percentage of female hatchlings to the population, the more northern and cooler nesting beaches from the northeast Florida to North Carolina nesting populations might mitigate that highly skewed female ratio by producing proportionally more male hatchlings [22]. For example, a study of the hatchling sex ratio for a North Carolina nesting beach determined a mean annual sex ratio of 58% female [46]. However, an investigation of a juvenile population of loggerheads in foraging habitat in North Carolina resulted in a more female-biased (75%) sex ratio [34]. Thus, one needs to consider the effects of climate change on the different nesting populations that contribute to the juvenile population when evaluating its sex ratio.

Conversely, a lack of a trend in our sex ratio data may indicate that loggerheads are adapting to warming temperatures. In fact, some have proposed that TSD might actually be an unexpected adaptation that could enable animals to survive warming effects of climate change [48]. For instance, nesting in different substrates, latitudes, or depths, or nesting during cooler time periods (at the beginning or end of the nesting season) could enable turtles to mitigate the harmful effects of increasing temperatures [46]. As an example, a ten day earlier median nesting date for a population of loggerheads nesting along Florida’s Atlantic coast from 1989 to 2003 was recorded [49]. Likewise, a male-biased population of red-eared sliders (Trachemys scripta elegans) was postulated to be the result of warming temperatures [50]. During the consequential longer nesting season, red-eared slider females laid an extra clutch when soil temperatures were relatively low, leading to the production of additional males [50]. Thus, changes in maternal behavior such as these could ameliorate some of the harmful environmental effects of climate change.

Animals with TSD can be viewed as the ‘canaries in the coal mine’ with respect to climate change [11], and these populations should be monitored to determine what effect, if any, climate change is having on sex ratios [13, 42, 51, 52], and ultimately, population viability. Many studies quantifying the effects of climate change on sea turtle population sex ratios have focused attention on monitoring hatchling sex ratios. However, considering the inherent intra- and inter-annual variation in hatchling sex ratios, the uncertainty associated with hatchling sex ratio estimation (i.e., estimates from small numbers of hatchlings or inferred from measured incubation temperatures or durations) [26], or the expense and logistical difficulty in sexing post-hatchling turtles [28], a more viable alternative would be to monitor changes in the sex ratio of the juvenile portion of the population [21, 29, 30]. Because this life stage integrates sex ratio variability over time [30, 31], allows for more random sampling of populations [21, 30], and can be sexed measuring blood testosterone levels that are verified via laparoscopy, monitoring effects of climate change at this life stage might more accurately reveal if mitigation measures to climate change are warranted [21, 32].

Despite this relevant management concern, long-term studies examining trends in secondary sex ratios for sea turtle populations are limited, with only two such studies having been conducted on juvenile loggerheads: in the Mediterranean Sea (necropsy of 271 turtles) [43] and in the northeast Atlantic (laparoscopy of 224 turtles with partial histological validation) [42]. Most other studies only look at a population’s sex ratio at a particular point in time. In contrast, our study is one of the first to examine sex ratios over a broad time span, encompassing ten years and sampling over 950 juvenile loggerhead turtles

Primary sex ratios for most loggerhead sea turtle populations, derived from nest temperatures or incubation durations [26], have been found to be strongly female-biased (see [53] for a review), with some populations in the western North Atlantic consisting of 90% or more females [53, 54]. Although still biased towards females, sex ratios of foraging juvenile loggerhead populations are not as highly skewed, displaying a 2F:1M ratio in Florida and Virginia [38], 3F:1M in North Carolina [34], 2F:1M in the northeast Atlantic [42], and 1.56F:1M in the Mediterranean [43]. However, operational sex ratios (estimated from adults actively breeding in a season) potentially appear to be more balanced [53] because male sea turtles breed more frequently than females and mate with multiple females, thus, providing a more balanced operational sex ratio despite a population’s highly skewed primary sex ratio.

Even with the existing female bias and its possible role in sea turtle life history, because sea turtles produce females during warm incubation temperatures [55], current predicted warming conditions have the potential to result in even more highly skewed sex ratios towards females, which in turn could lead to reduced genetic viability [56] and extinction [9, 57]. In a study of the potential impacts of climate change on loggerhead nesting populations, modeling exercises [46] showed that even the minimum predicted increase in air temperature (2°C) in nesting areas which are currently highly female-biased (such as southern Florida) would result in total feminization of many nests while a 3°C increase would result in many of these nests experiencing lethal incubation temperatures. However, given other environmental factors that can influence incubation temperatures (e.g., rainfall), even under an increasing temperature scenario, there will likely still be times when beach temperatures are male-producing [26].

Although sea turtles have survived past geologic temperature fluctuations, the methods used to cope with past changes and the speed with which those changes occurred is not known [58]. Current predicted climate changes are projected to happen at relatively rapid rates [5], and little empirical knowledge about the ability of sea turtles to survive these changes is available. If warming global temperatures result in detrimental changes to population sex ratios, animals with TSD could react in a number of ways–with an evolutionary response by modifying pivotal temperatures or changing to genotypic sex determination, an ecological response by altering geographic ranges or timing of nesting, or by becoming extinct [8]. Sea turtle populations are currently imperiled due to a number of anthropogenic threats including habitat alteration and loss, pollution, and incidental capture in fisheries [59]. If sea turtles are not able to adjust to impending climate changes, the result could be detrimental. Therefore, long-term monitoring of sea turtle populations should continue; however, studies should focus more on investigation of secondary, rather than primary, sex ratios, so that accurate assessments can detect changes in time for mitigation measures to be effective.

Supporting Information

S1 Table. Summary of turtle testosterone level and sex determination.

https://doi.org/10.1371/journal.pone.0160911.s001

(DOCX)

Acknowledgments

The authors gratefully acknowledge the efforts of many individuals during this 10 year project: L. Evich, G. Blanvillain, M. Lee, and R. Patterson for conducting radioimmunoassays; B. Chittick, G. Lewbart, A. Segars, M. A. Stamper, S. Weber, and S. Willens for conducting laparoscopies; the many volunteers for their assistance with field work and laparoscopies, most especially R. Boettcher, J. Bybee, W. Cluse, T. Ellis, S. P. Epperly, M.H. Godfrey, K. Hart, T. Kelly, M. Landon, Y. Levy, J. Lynch, C. McClellan, N. Mihnovets, M. Snover, W. Teas, H. Valentine, J. Weaver and the North Carolina State University College of Veterinary Medicine students; A.Schueller for statistical advice. The manuscript benefited from reviews by C. Allen, A. Chester, M. H. Godfrey, A. Hohn, P. Marraro, A.Schueller, and anonymous reviewers. Finally, we are indebted to the numerous pound net fishermen for allowing us access to sea turtles incidentally captured in their nets. The Owens lab effort is Contribution number 477 of the Grice Marine Laboratory, College of Charleston. This research was conducted under the authority of U.S. Endangered Species Act Section 10(a)(1)(A) scientific research permits from the U.S. Fish and Wildlife Service (#TE-676379) and the NMFS (#1260). The NMFS does not approve, recommend, or endorse any product or material mentioned in this publication.

Author Contributions

  1. Conceptualization: DWO LA.
  2. Data curation: JBM AGH.
  3. Formal analysis: JBM.
  4. Investigation: JBM LA AGH LRG CAH.
  5. Methodology: DWO.
  6. Project administration: JBM.
  7. Resources: DWO.
  8. Supervision: LA.
  9. Writing – original draft: JBM.
  10. Writing – review & editing: JBM LA AGH LRG CAH DWO.

References

  1. 1. Levitus S, Antonov JI, Wang JL, Delworth TL, Dixon KW, Broccoli AJ. Anthropogenic warming of Earth's climate system. Science. 2001;292(5515):267–70. pmid:ISI:000168074000041.
  2. 2. Brohan P, Kennedy JJ, Harris I, Tett SFB, Jones PD. Uncertainty estimates in regional and global observed temperature changes: A new data set from 1850. Journal of Geophysical Research. 2006;111. D12106 pmid:ISI:000238599200001.
  3. 3. Hansen J, Sato M, Ruedy R, Lo K, Lea DW, Medina-Elizade M. Global temperature change. Proceedings of the National Academy of Sciences. 2006;103(39):14288–93.
  4. 4. Walther GR, Post E, Convey P, Menzel A, Parmesan C, Beebee TJC, et al. Ecological responses to recent climate change. Nature. 2002;416(6879):389–95. pmid:ISI:000174607800036.
  5. 5. IPCC. Climate Change 2014: Synthesis Report. Contribution of Working Groups I, II, and III to the Fifth Assessment Report of the Intergovernmental Panel on Climate Change. IPCC, Geneva, Switzerland: 2014.
  6. 6. Hughes LI. Biological consequences of global warming: is the signal already apparent? Trends in Ecology & Evolution. 2000;15(2):56–61. pmid:10652556.
  7. 7. Parmesan C, Yohe G. A globally coherent fingerprint of climate change impacts across natural systems. Nature. 2003;421(6918):37–42. pmid:ISI:000180165500029.
  8. 8. Janzen FJ, Paukstis GL. Environmental sex determination in reptiles: ecology, evolution, and experimental design. Q Rev Biol. 1991;66(2):149–79. pmid:1891591.
  9. 9. Janzen FJ. Climate change and temperature-dependent sex determination in reptiles. Proceedings of the National Academy of Sciences. 1994;91(16):7847–490. pmid:8052608; PubMed Central PMCID: PMCPMC44426.
  10. 10. Yntema CL, Mrosovsky N. Sexual differentiation in hatchling loggerheads incubated at different controlled temperatures. Herpetologica. 1980;36:33–6.
  11. 11. Mitchell NJ, Janzen FJ. Temperature-dependent sex determination and contemporary climate change. Sex Dev. 2010;4(1–2):129–40. pmid:ISI:000276343400013.
  12. 12. Mitchell NJ, Allendorf FW, Keall SN, Daugherty CH, Nelson NJ. Demographic effects of temperature-dependent sex determination: will tuatara survive global warming? Global Change Biology. 2010;16(1):60–72. pmid:ISI:000274419200006.
  13. 13. Hamann M, Godfrey MH, Seminoff JA, Arthur K, Barata PCR, Bjorndal KA, et al. Global research priorities for sea turtles: informing management and conservation in the 21st century. Endangered Species Research. 2010;11(3):245–69.
  14. 14. Patino-Martinez J, Marco A, Quinones L, Hawkes LA. A potential tool to mitigate the impacts of climate change to the caribbean leatherback sea turtle. Global Change Biology. 2012;18(2):401–11. pmid:ISI:000299042500001.
  15. 15. Fuentes MMPB, Hamann M, Limpus CJ. Past, current and future thermal profiles of green turtle nesting grounds: Implications from climate change. Journal of Experimental Marine Biology and Ecology. 2010;383(1):56–64.
  16. 16. Laloë J-O, Esteban N, Berkel J, Hays GC. Sand temperatures for nesting sea turtles in the Caribbean: Implications for hatchling sex ratios in the face of climate change. Journal of Experimental Marine Biology and Ecology. 2016;474:92–9. http://dx.doi.org/10.1016/j.jembe.2015.09.015.
  17. 17. Fuentes MMPB, Porter WP. Using a microclimate model to evaluate impacts of climate change on sea turtles. Ecological Modelling. 2013;251(0):150–7. http://dx.doi.org/10.1016/j.ecolmodel.2012.12.020.
  18. 18. Saba VS, Stock CA, Spotila JR, Paladino FV, Santidrian Tomillo P. Projected response of an endangered marine turtle population to climate change. Nature Climate Change Letters. 2012.
  19. 19. Witt MJ, Hawkes LA, Godfrey MH, Godley BJ, Broderick AC. Predicting the impacts of climate change on a globally distributed species: the case of the loggerhead turtle. Journal of Experimental Biology. 2010;213(6):901–11. Epub 2010/03/02. pmid:20190115.
  20. 20. Mrosovsky N, Kamel SJ, Diez CE, van Dam RP. Methods of estimating natural sex ratios of sea turtles from incubation temperatures and laboratory data. Endangered Species Research. 2009;8:147–55.
  21. 21. Wibbels TR. Critical approaches to sex determination in sea turtles. In: Lutz PL, Musick JA, Wyneken J, editors. The Biology of Sea Turtles Volume II. 2nd. Boca Raton, Florida: CRC Press; 2003. p. 103–34.
  22. 22. Mrosovsky N, Hopkins-Murphy SR, Richardson JI. Sex ratio of sea turtles: Seasonal changes. Science. 1984;225:739–41. pmid:17810293
  23. 23. Mrosovsky N. Sex ratios of sea turtles. Journal of Experimental Zoology. 1994;270:16–27.
  24. 24. Laloe J-O, Cozens J, Renom B, Taxonera A, Hays GC. Effects of rising temperature on the viability of an important sea turtle rookery. Nature Climate Change. 2014;4(6):513–8.
  25. 25. Broderick AC, Godley BJ, Hays GC. Metabolic Heating and the Prediction of Sex Ratios for Green Turtles (Chelonia mydas). Physiological and Biochemical Zoology. 2001;74(2):161–70. pmid:11247735
  26. 26. Wyneken J, Lolavar A. Loggerhead Sea Turtle Environmental Sex Determination: Implications of Moisture and Temperature for Climate Change Based Predictions for Species Survival. J Exp Zool Part B. 2015;324(3):295–314. pmid:WOS:000353838800010.
  27. 27. Wyneken J, Epperly S, Heppell SS, Crowder L, editors. The importance of annual variation in understanding sea turtle sex ratios. Twenty-sixth annual symposium on sea turtle biology and conservation, Book of Abstracts; 2006; Athens, Greece: International Sea Turtle Symposium.
  28. 28. Wyneken J, Epperly SP, Crowder LB, Vaughan J, Esper KB. Determining sex in posthatchling loggerhead sea turtles using multiple gonadal and accessory duct characteristics. Herpetologica. 2007;63(1):19–30.
  29. 29. Shoop CR, Ruckdeschel CA, Kenney RD. Female-biased sex ratio of juvenile loggerhead sea turtles in Georgia. Chelonian Conservation and Biology. 1998;3:93–6.
  30. 30. Wibbels TR, Owens DW, Morris YA, Amoss MS. Sexing techniques and sex ratios for immature loggerhead sea turtles captured along the Atlantic coast of the United States. 1987.
  31. 31. Wibbels TR, Martin RE, Owens DW, Amoss MSJ. Female-biased sex ratio of immature loggerhead sea turtles inhabiting the Atlantic coastal waters of Florida. Canadian Journal of Zoology. 1991;69:2973–7.
  32. 32. Wibbels TR, Owens DW, Limpus CJ. Sexing juvenile sea turtles: is there an accurate and practical method? Chelonian Conservation and Biology. 2000;3(4):756–61.
  33. 33. Epperly SP, Braun-McNeill J, Richards PM. Trends in catch rates of sea turtles in North Carolina, USA. Endangered Species Research. 2007;3:283–93.
  34. 34. Braun-McNeill J, Epperly SP, Owens DW, Avens L, Williams E, Harms CA. Seasonal reliability of testosterone radioimmunoassay (RIA) for predicting sex ratios of juvenile loggerhead (Caretta caretta) turtles. Herpetologica. 2007;63(3):275–84. pmid:WOS:000250133300004.
  35. 35. Higgins E, Pearson JC. Examination of the summer fisheries of Pamlico and Core Sounds, N.C., with special reference to the destruction of the undersized fish and the protection of the gray trout Cynoscion regalis (Block and Schneider), Appendix II. Report of the United States Commissioner of Fisheries for the Fiscal Year 1927 with Appendices. Government Printing Office, Washington D.C.: US Department of Commerce, Bureau of Fisheries; 1928. p. 29–65.
  36. 36. Guthrie JF, Kroger RL, Gordy HR, Lewis CW. The long-haul fishery of North Carolina. Marine Fisheries Review. 1973;35(12):27–33.
  37. 37. Owens DW, Ruiz GJ. New methods of obtaining blood and cerebrospinal fluid. Herpetologica. 1980;36:17–20.
  38. 38. Owens DW. Hormones in the life history of sea turtles. In: Lutz PL, Musick JA, editors. The Biology of Sea Turtles. Boca Raton, Florida: CRC Press; 1997. p. 315–41.
  39. 39. Owens DW, Hendrickson JR. Endocrine studies and sex ratios of the green sea turtle, Chelonia mydas Florida Marine Research Publication. 1978;33:12–4.
  40. 40. Wyneken J. The Anatomy of Sea Turtles. US Department of Commerce NOAA Technical Memorandum NMFS-SEFSC-470. 2001. p. 1–172.
  41. 41. Zar JH. Biostatistical Analysis. 4th ed. Upper Saddle River, New Jersey 07458: Prentice-Hall, Inc.; 1999. 663 p.
  42. 42. Delgado C, Canário AVM, Dellinger T. Sex ratios of loggerhead sea turtles Caretta caretta during the juvenile pelagic stage. Marine Biology. 2010;157(5):979–90.
  43. 43. Maffucci F, D’Angelo I, Hochscheid S, Ciampa M, Martino G, Travaglini A, et al. Sex ratio of juvenile loggerhead turtles in the Mediterranean Sea: is it really 1:1? Marine Biology. 2013;160(5):1097–107.
  44. 44. Scott R, Marsh R, Hays GC. Life in the really slow lane: loggerhead sea turtles mature late relative to other reptiles. Functional Ecology. 2012;26(1):227–35. pmid:ISI:000299204300025.
  45. 45. Avens L, Goshe LR, Coggins L, Snover ML, Pajuelo M, Bjorndal KA, et al. Age and size at maturation- and adult-stage duration for loggerhead sea turtles in the western North Atlantic. Marine Biology. 2015;186:1–19.
  46. 46. Hawkes LA, Broderick AC, Godfrey MH, Godley BJ. Investigating the potential impacts of climate change on a marine turtle population. Global Change Biology. 2007;13(5):923–32.
  47. 47. Bass AL, Epperly SP, Braun-McNeill J. Multi-year analysis of stock composition of a loggerhead turtle (Caretta caretta) foraging habitat using maximum likelihood and Bayesian methods. Conservation Genetics. 2004;5:783–96.
  48. 48. Kallimanis AS. Temperature dependent sex determination and climate change. Oikos. 2010;119:197–200.
  49. 49. Weishampel JF, Bagley DA, Ehrhart LM. Earlier nesting by loggerhead sea turtles following sea surface warming. Global Change Biology. 2004;10:1424–7.
  50. 50. Tucker JK, Dolan CR, Lamer JT, Dustman EA. Climatic warming, sex ratios, and red-eared sliders (Trachemys scripta elegans) in Illinois. Chelonian Conservation and Biology. 2008;7(1):60–9. pmid:ISI:000258608800009.
  51. 51. Hawkes LA, McGowan A, Godley BJ, Gore S, Lange A, Tyler CR, et al. Estimating sex ratios in Caribbean hawksbill turtles: testosterone levels and climate effects. Aquatic Biology. 2013;18(1):9–19.
  52. 52. Wyneken J, Lolavar A. Loggerhead sea turtle environmental sex determination: Implications of moisture and temperature for climate change based predictions for species survival. Journal of Experimental Zoology (Molecular and Developmental Evolution). 2015;324(3):295–314. pmid:ISI:000353838800010.
  53. 53. Hays GC, Schofield G, Mazaris AD. Different male versus female breeding periodicity helps mitigate offspring sex ratio skews in sea turtles. Frontiers in Marine Science. 2014;http://dx.doi.org/10.3389/fmars.2014.00043.
  54. 54. Freedberg S, Wade MJ. Cultural inheritance as a mechanism for population sex-ratio bias in reptiles. Evolution. 2001;55(5):1049–55. pmid:11430641
  55. 55. Standora EA, Spotila JR. Temperature dependent sex determination in sea turtles. Copeia. 1985;1985(3):711–22.
  56. 56. Tomillo PS, Oro D, Paladino FV, Piedra R, Sieg AE, Spotila JR. High beach temperatures increased female-biased primary sex ratios but reduced output of female hatchlings in the leatherback turtle. Biological Conservation. 2014;176:71–9.
  57. 57. Hulin V, Delmas V, Girondot M, Godfrey MH, Guillon JM. Temperature-dependent sex determination and global change: are some species at greater risk? Oecologia. 2009;160(3):493–506. Epub 2009/03/12. pmid:19277719.
  58. 58. Hamann M, Limpus CJ, Read MA. Vulnerability of marine reptiles in the Great Barrier Reef to climate change. In: Johnson JE, Marshall PA, editors. Climate Change and the Great Barrier Reef: A Vulnerability Assessment. Australia: Great Barrier Reef Marine Park Authority and Australian Greenhouse Office; 2007. p. 466–96.
  59. 59. Lutcavage ME, Plotkin PT, Witherington BE, Lutz PL. Human impacts on sea turtle survival. In: Lutz PL, Musick JA, editors. The Biology of Sea Turtles. Boca Raton, Florida: CRC Press; 1997. p. 387–409.