Wyneken, Jeanette

Sea turtle nest success, defined as the number of eggs in a nest that successfully hatch and emerge, is closely linked to environmental conditions. Interacting biotic and abiotic factors influence hatching and hatchling emergence success. To date, combinations of multiple factors interacting together, which result in highly successful sea turtle nests are not well understood. Using 25-years of historic nest data and local expert experience, I identified five historically successful loggerhead (Caretta caretta) nesting beaches (hotspots) along the Florida (USA) Atlantic coast and measured nest environments along with nest success. Principal component analysis was used to reduce 12 environmental variables so that the relative contributions of sand characteristics, nest temperature, sand moisture, and nest location were considered. The nest environments differed among nesting beaches and were broadly segregated into two distinct climates: subtropical (hot and humid) and warm-temperate (warm and dry). I found that nests at subtropical sites, compared with the warm-temperate sites, were characterized by environmental gradients in contrasting ways. Nest locations were predominantly mid-beach in subtropical sites but clustered at higher elevations and closer to the base of the dune at warm-temperate climate sites. Collectively, highly successful nest hotspots represent a mosaic of abiotic factors providing conditions that promote successful hatching and emergence. This new perspective on consistently successful loggerhead nesting beach traits demonstrate that the key traits of sea turtle nesting habitat vary with prevailing climate type and should be managed accordingly.

Despite decades of conservation efforts, population recovery remains elusive for the loggerhead sea turtles (Caretta caretta) nesting in Florida, the largest aggregation globally. Limited studies exist regarding reproductive strategies and in-water habitat use of loggerheads in southeastern Florida. I used satellite telemetry to track the movements of 17 nesting loggerheads on Juno and Jupiter Beaches during the 2020 and 2021 nesting seasons. The majority of females displayed high nest-site fidelity. Inter-nesting intervals ranged from 10–19 days and were negatively correlated with water temperatures. Core inter-nesting areas ranged from 3.7–805.8 km2 and were located a mean 1.6 km from land. Mean clutch frequency was 5.9 nests/female, the highest reported for any loggerhead population worldwide. These findings suggest the number of females in the southeastern Florida population may be over-estimated due to an underestimated clutch frequency. Protective measures should target high-use coastal areas to maximize conservation benefits.

The eggs of all sea turtle species develop in underground nests on oceanic nesting beaches. Eggs are unattended and their incubation conditions are subject to effects of the environment. Nest temperature influences various aspects of hatchling biology, including sex determination. Past studies identified that sea turtle embryos have a warm female cool male response pattern and rainfall has been thought to cool nest temperature. The effects of rainfall or periods of drought were often inferred but not verified. Using laboratory and field studies, I examined how changes in environmental factors during incubation, particularly sand moisture, can affect nest conditions and hatchling biology. I derived temperature-sex ratio response curves for eggs incubated at different moisture levels to determine the effect of moisture on how embryos respond to temperature. I also studied how increasing moisture levels in relocated nests through daily watering influence nest conditions and discuss if this method is an effective mitigation strategy for the detrimental effects of increasing temperatures on embryo survival and sex ratios. I investigated how environmental factors, nest conditions, and hatchling biology can differ among sites on a nesting beach. Extreme moisture conditions, both low and high, result in a narrower transition between one sex ratio bias to another. I demonstrated that watering nests decreases nest temperatures and increases hatching success but watering has a minimal impact on sex ratios. Ambient beach conditions vary slightly in air temperature, rainfall, solar radiation, and humidity, depending on beach location. Nest conditions such as nest temperature and moisture also differ, but hatching success and sex ratios do not vary among different sites on the same nesting beach in Boca Raton, Florida. Ultimately, these studies together help identify and demonstrate how these environmental factors and drivers can affect the nest environment during incubation. Further developing our understanding of environmental factors, particularly nest moisture, and their variability will provide better predictions of future climate change effects and perhaps create more effective mitigation strategies.

Currently, one of the most critical research priorities in wildlife science is to understand, describe, and predict how the unprecedented rate of climate change will impact organisms and ecosystems. This is particularly essential for thermally sensitive organisms that are already imperiled, such as turtles.
For all known turtle species, the nest incubation environment plays a critical role in many developmental processes which can directly influence a number of phenotypic traits, such as body size, mass, locomotor performance, behavior and even sex. Most chelonians (and all extant marine turtles) possess a mechanism known as temperature dependent sex determination (TSD), whereby gonads differentiate into ovaries or testes depending on the incubation temperature of the eggs during a critical period of embryonic development. The rapid rate of climate change highlights the need for a clear understanding of how potential changes in the nest environment will affect turtle development and hatchling phenotype. However, it is poorly understood how different environmental factors interact with the embryo’s own genetic program to produce a specific phenotype. My thesis aims to (i) provide a better understanding of the complex relationship between the developing embryo and the nest environment and its effect on hatchling phenotype, and (ii) offer a solution to the difficulties associated with identifying primary sex ratios in turtle species with TSD.

Sea turtle hatchlings emerge from their nest and quickly crawl to the surf. During the crawl, hatchlings may encounter threats, biotic and abiotic, which can affect their ability to successfully reach the surf. The impact of these threats on hatchling survival during that crawl is largely undocumented. Current methods used to estimate cohort recruitment rely heavily on nest inventory data. This method, however, does not account for post-emergent hatchling mortality that occurs during the crawl. During the 2017-2018 nesting seasons, I quantified the fates of 1,379 loggerhead (Caretta caretta) hatchlings from 26 nest emergences during their crawl from the nest to the surf on the east and west coasts of Florida. I documented hatchling fates at 5 Florida nesting beaches: Wabasso, Boca Raton, Keewaydin Island, Naples, and Anna Maria Island. Overall, 6.5% of all emergent hatchlings died during the crawl from the nests to the surf. Ghost crabs, night herons, foxes, and coyotes killed hatchlings and photopollution and barriers on the beach (both abiotic threats) caused hatchling mortality. Anthropogenic (abiotic) threats accounted for more mortality than did predators. In order to assess how beach urbanization impacts hatchling mortality, I categorized each study site as urban (Wabasso and Naples), intermediate (Anna Maria Island and Boca Raton), or natural (Keewaydin Island) based on the relative levels of shoreline development and human activity at each beach. Sites with intermediate levels of urbanization accounted for greater levels of hatchling mortality than at other beaches due to the absolutely larger numbers of hatchlings lost to a disorientation event and to a beach barrier. Given the small numbers of emergences, at all sites, only a small proportion of the hatchlings mortalities (e.g., between 3 and 12 percent), site type could not be rigorously used as a discriminator. My results provide a better understanding of how specific environmental threats contribute to hatchling mortality. While nest-to-surf mortality is relatively low, its cumulative costs add up to several hundreds of thousands of hatchlings. Armed with this information, nesting beach managers can assess risks and focus their efforts to implement the most effective management practices to minimize losses of this imperiled species.

The reproductive behavior of migratory organisms is difficult to characterize as
the mating behavior can be difficult to observe. For some species, one sex can be readily
observable, but the other may remain hidden, confounding attempts to assess population
demographics. For such species, it can be difficult to determine the sex ratio of the
population. Without accurate accounts of the numbers of males and females, conservation
methods may be insufficient and their performance unclear. Alternative methods of
measuring sex ratios therefore must be used to estimate the number of individuals and
assess breeding behavior. Here I identified breeding sex ratios (BSR) measured using
paternity analysis of offspring through exclusion analysis to quantify the numbers of
males contributing. Here I discuss the mating behavior of three species of marine turtle
that nest in southern Florida: the loggerhead (Caretta caretta), the leatherback (Dermochelys coriacea), and the green sea turtle (Chelonia mydas) at three beaches
(Boca Raton, Juno Beach, and Sanibel Island) from 2013-2017; over 400 nesting females
were identified and genotyped and almost 7,000 hatchlings were collected and
genotyped. Females from all three species successfully mated with more than one male in
all years analyzed demonstrating multiple paternity of clutches. For loggerheads, many
male genotypes were identified, suggesting that females likely mate en route to their
nesting grounds, inducing a male-biased BSR. Examination of females that were sampled
more than once per season (repeats), evidence of sperm storage was found for all nests
and some turtles might mate in between nesting events. Leatherback females displayed a
higher rate of multiple paternity than was previously published for other Caribbean
nesting sites; I hypothesize that this result may be due to a mainland nesting beach effect.
The leatherback BSR over all years was approximately 1:1, and one male was identified
fathering than two different females’ nests (polygyny). For green turtles, multiple
paternity was found and there was evidence of polygyny. Across all three species,
evidence for indirect benefits of mating multiply (hatching success or larger hatchlings)
was weak or not supported. Together, the four studies contribute to the overall body of
reproductive behavior studies.

Sound assessment of the status of a threatened or endangered organism depends on understanding
key aspects of behavior throughout its life history. Sometimes organisms can be difficult to observe and
key aspects of behavior may not be accessed directly. Alternative assessment techniques include using
molecular markers to identify fundamental relationships among males and females. In the context of
assessing the status of imperiled populations’ sex ratios, population size and the relatedness of the
individuals are important metrics. Environmental sex determination directs developing marine turtle sex
so that primary sex ratios depend upon weather and climate; those sex ratios are estimated by proxies.
Adult population sizes are inferred from numbers of females nesting on the beach, but numbers of
males are unknown. Male breeding population size can be estimated from subtracting maternal
genotypes from genotypes of offspring exclusion analyses. The resulting adult sex ratios differ greatly
from those estimated for hatchlings. To refine current adult sex ratios in ways that are relevant to
production of future generations and add to our understanding of effective population size we compare
the breeding sex ratios the number of males and females contributing to a population of three species
of sea turtles nesting in southern Florida. We will use the same genetic data to measure relatedness of
the female nesters and the male contributors and describe how that relates to genetic flow and
population structure.

Nest sand temperature strongly influences development of sea turtle embryos and sex differentiation;
however in nature eggs experience temperature along with other environmental factors. We tested the
hypothesis that moisture effects sea turtle hatchling sex ratios. We studied the relationships among
moisture, temperature, and loggerhead Caretta caretta sex ratios in an experimental study. Eggs were incubated in sterile nest sand in the laboratory under different moisture regimes to test the role of
humidity at a constant incubation temperature. Incubator temperature was set at 29.4 degrees C, a
temperature that is slightly above the temperature that should yield a 1:1 sex ratio. Nest moisture was
maintained by daily DI water treatments and high relative humidity was maintained with the aid of a mist
humidifier throughout incubation. All hatchlings were collected, raised for several months and sexed
laparoscopically to establish sex ratios for each treatment. The experimental treatments tested the
effects of i very high moisture, ii moisture with potential for evaporative cooling, and iii moisture added
at average rain temperatures plus the potential for evaporative cooling. The nests were expected to
produce a moderate female bias if moisture played no role. We found 87-96 males across all
experimental treatments. Our results support our hypothesis. High moisture conditions can produce
shifts in developmental response from that
expected based on temperature alone.

Marine turtles exhibit temperature-dependent sex determination (TSD). During critical periods of
embryonic development, the nest’s thermal environment directs whether an embryo will develop as a
male or a female. At warmer sand temperatures the nest tends to produce female-biased sex ratios.
The rapid increase of global temperature highlights the need for a clear assessment of effects on sea
turtle sex ratios. However, identifying hatchling sex ratios at rookeries remain coarse estimates due to
the lack of any external gender markers. We rely mainly upon laparoscopic procedures to verify a
hatchling sex; however, in some species, morphological sex can be ambiguous even at the histological
level. Recent studies using immunohistochemical techniques identified that red-eared slider
(Trachemys scripta) embryos over-expressed a particular cold-induced RNA binding protein in the
ovaries in comparison to the testes. This principle allows the distinction between females and males.
We developed a variation of this technique and successfully identified the sexes of loggerhead sea
turtle (Caretta caretta) hatchlings, as confirmed by standard histological and laparoscopic methods that
reliably identifies the sex in this species. Next, we tested a more challenging species, the leatherback
turtle (Dermochelys coriacea), which retains many neotenic features. The morphology of leatherback
hatchling gonads remains difficult to interpret, particularly when dead-in-nest hatchlings and embryos
are the source tissues. In summary, this new and more efficient technique enhances our ability to investigate and identify baseline hatchling sex ratios, a critical progression in assessing global climate
change on sea turtle populations.