Impact of Intrabeach Incubation Substrate Variability on Sea Turtle Reproductive Success
ABSTRACT
Variation in sand composition at the individual beach level is an important factor for hatching success and hatchling phenotype that does not appear to have been previously investigated. We studied variation in intrabeach substrate composition in an important loggerhead turtle (Caretta caretta) nesting colony on the island of Maio, Cabo Verde, and examined its relationship with embryonic development success, hatchling phenotype, and hatchling vitality. Thirty-two nests from different females were studied under experimental conditions, controlling for beach position, depth, humidity, and temperature in a randomized block design. Two sand treatments corresponding to 2 types of sand from the same beach were used. While nesting females selected nesting sites indiscriminately between substrate types, their nests differed significantly in all variables measured for reproductive success (hatching success, hatchling size, and hatchling agility) between the 2 sand types. Therefore, the intrabeach observations of substrate phenotype of progeny found here could differentially affect offspring survival. The effects of choosing different nesting sites will generally act to spread the probability of selective pressures on embryos. Variation in reproductive success was due to differences between substrate types within the beach rather than the location of nests within the beach. The importance of determining sand types with the highest hatching success, even within the same beach, should be greatest in colonies or populations highly threatened with extinction, where the hatching success of each nest becomes of utmost importance.
Sea turtles are ectothermic, long-lived, late-maturing reptiles, well adapted to life in water. Their primary connection to the terrestrial environment is the sandy beaches used for nesting (Ackerman 1980; Miller 1997). Adult females exhibit natal philopatry, returning to their natal areas and choosing the same beach or nearby beaches to lay their eggs (Hulin and Guillon 2007; Stiebens et al. 2013; Brothers and Lohmann 2015; Clusa et al. 2018; Patino-Martinez et al. 2022a). Because the eggs do not receive parental care, nest site selection plays a crucial role in determining environmental conditions for incubation (Miller et al. 2003).
The combination of some biotic factors—such as microorganisms (Phillott and Parmenter 2001; Craven et al. 2007; Keene et al. 2014; Sarmiento-Ramírez et al. 2014), roots and predators (Conrad et al. 2011; Marco et al. 2015), and other abiotic factors such as temperature (Fisher et al. 2014; Booth 2017), water potential (McGehee 1990; Caut et al. 2010; Patino-Martínez et al. 2014), substrate composition (Mortimer 1990; Foley et al. 2006; Fadini et al. 2011; Marco et al. 2017; Tacchi et al. 2019), and the concentration of available gases (Garrett et al. 2010; Cheng et al. 2015)—determine embryonic developmental success (Wallace et al. 2004; Garrett et al. 2010), hatching success, and the sex and phenotype of hatchlings (Matsuzawa et al. 2002; Glen et al. 2003; Patrício et al. 2017).
As clutch survival may vary according to substrate type, e.g., light sand beaches of biogenic origin have higher hatching success than dark beaches of volcanic origin and higher absorbance (Patino-Martínez et al. 2022b), nest site selection can determine the survival of nests (Hawkes et al. 2010; Patrício et al. 2018). The effect of nesting substrate heterogeneity on sea turtle reproductive success has been studied comparing beaches (Mortimer 1990; Fadini et al. 2011), colonies (Marco et al. 2017; Saito et al. 2019; Tacchi et al. 2019; Patino-Martinez et al. 2022b), and species (Saito et al. 2019; Suhaimi et al. 2020). However, this study examines, for the first time, the possible effect of intrabeach variation in incubation substrate on nest success and fitness of loggerhead turtle hatchlings.
This study was conducted in the volcanic archipelago of Cabo Verde, located 500 km from Senegal in the Atlantic Ocean. Cabo Verde is home to what may be the largest loggerhead turtle (Caretta caretta) nesting subpopulation in the world (Patino-Martínez et al. 2021). The loggerhead turtle is classified by the International Union for Conservation of Nature as globally “Vulnerable” and regionally “Endangered” due to the ongoing decline in the area, extent, and/or quality of its habitat (Casale and Tucker 2017). This subpopulation has been identified as a separate genetic stock (Monzon-Arguello et al. 2010; Wallace et al. 2010) with multiple nesting groups within the colony (Stiebens et al. 2013; Baltazar-Soares et al. 2020). The island of Maio hosts a significant proportion of annual nests from this subpopulation, with 4063–46,160 (mean 15,440) annual nests between 2016 and 2022. This study compared sand from 2 areas of the same beach and assessed the following: 1) grain size, composition, and colorimetry; 2) nesting success; 3) embryonic development success; 4) hatching success; and 5) hatchling body condition. Understanding nesting behavior and its consequences for reproductive success within and between beaches can support the conservation of endangered species.
METHODS
Study Area. — Fieldwork was conducted on the island of Maio, Cabo Verde (Fig. 1; Praiona Beach; lat 15°16ʹ5.2″N, long 23°6ʹ21.3″W), from August to October 2018. Praiona Beach measures 613 m in length, has an average slope of 13°, and varies in width between 8 and 27 m from the low tide line to the dune vegetation (Zygophyllum waterlotii and Suaeda vermiculata). It is an undeveloped and undisturbed beach, free from light pollution and with minimal human activity. For this study, the beach was divided into 2 sectors from north to south (300 m apart), which were distinguished by differences in sand color and grain size (CLN = coarse, light sand, northern; FDS = fine, dark sand, southern; see Fig. 1C).
Citation: Chelonian Conservation and Biology: Celebrating 25 Years as the World’s Turtle and Tortoise Journal 22, 2; 10.2744/CCB-1581.1
Nesting Success. — All nesting activities were recorded over 80 nights (August to October 2018) in both beach zones between 2000 hrs and 0600 hrs. All nesting activities, including nesting attempts resulting in nesting or no nesting, were recorded and marked to avoid duplication. The percentage of nesting attempts that resulted in successful nesting was calculated for each study area.
Intrabeach Substrate Analysis. — For particle-size analysis, 32 sand samples (approximately 300 g, 45-cm depth) were collected from each beach treatment (CLN = 16 and FDS = 16; Fig. 1C). Each sample was sieved according to the Atterberg grain size scale (Seed et al. 1964). The granules were separated into 2 size classes: fine (< 0.5 mm) and coarse (≥ 0.5 mm). The percentage of granulometric fractions (Fr) was calculated by dividing the weight of each fraction (Pfraction) by the total weight of the sample (Ptotalsample): Fr = Pfraction/Ptotalsample × 100%. The sand color of the 2 beach areas was classified using the Munsell color chart system (Fan et al. 2017).
Experimental Study. — To evaluate the influence of intrabeach substrate variation on nest incubation under standardized conditions, we conducted an experimental study using 32 nests. The nests were incubated under 2 different sand treatments (CLN and FDS). To minimize the effects of seasonality and nest position on hatching success, 16 blocks consisting of the 2 treatments were evenly distributed between the 2 zones (8 blocks per zone). In each block (Fig. 2A), 2 nests from the same beach were placed 2.5 m apart, at the same distance from the high tide line, and on the same date. Clutches were relocated using new plastic bags, with a maximum time of 120 min between oviposition and relocation.
Citation: Chelonian Conservation and Biology: Celebrating 25 Years as the World’s Turtle and Tortoise Journal 22, 2; 10.2744/CCB-1581.1
For each experimental nest, a cylindrical chamber (1 m in diameter and 0.8 m deep) was excavated and filled with dry sand from the corresponding treatment (CLN or FDS). The sand was then moistened from the surface with 15 l of seawater 5 d before nest relocation. The relocated nests were buried 45 cm deep in a vase-shaped nest chamber within the experimental cylinder. Root debris was removed from the study substrates and nests were monitored daily throughout incubation.
Sand moisture was measured at a depth of 45 cm at the beginning (day 0) and end (day 53) of incubation in each of the study nests (n = 64, 300 g/sample). Sand incubation temperature was recorded at a 45-cm depth at 4 points (2 per zone) using Hobo Stow Away TidbiT v2 Onset thermometers with an accuracy of ± 0.2°C. Simultaneous recordings were obtained every 30 min throughout the experiment. Thermometers were checked for accuracy (deviation less than 0.2°C) by placing them in a box with a known temperature at least 48 hrs before and after the experimentation.
Hatchling Body Condition. — During emergence, nests were monitored daily, and all emerging hatchlings were counted (hatchlings were retained by half-buried plastic net cylinders: 50-cm diameter and 45-cm height on the sand surface). To assess the influence of intrabeach substrate variation on hatchling morphology and locomotion, a random sample of 167 hatchlings (CLN = 96, FDS = 71) was selected from all hatched nests for biometric analysis. All hatchlings were weighed using a digital scale (Smart Weigh Digital Pro Pocket Scale TOP2kg, accuracy ± 0.1 g), and measured (carapace length and maximum carapace width) using a hand-held caliper (BM-RS/150 mm, accuracy ± 0.05 mm). Hatchling performance was measured as the time taken to turn from the “face up” position to the normal “face down” run position (self-righting ability). These tests were conducted outdoors when the hatchlings were active on moist, compact sand. Each test was repeated 3 times per individual, and the results were averaged. In this study, the potential effects of circadian rhythm on hatchling activity were not standardized. After the physical tests, the hatchlings were immediately released into the sea. All unhatched eggs were dissected 72 hrs after the last nest emergence. Embryo development was categorized as follows: 0 = undeveloped, 1 = early embryonic death, 2 = middle embryonic death, and 3 = late embryonic death. Embryonic development success was calculated as the percentage of eggs that reached the final stage of incubation (category 3), and hatching success was calculated as the percentage of eggs that hatched (category 4).
Data Analysis. — All data were checked for normality using the Shapiro-Wilk test and for homogeneity of variance using Levene’s test. When necessary, data were transformed as log(x + 1) to ensure normality. The difference in mean grain size between the 2 beach zones was assessed using the parametric Student t test for independence. The difference in nesting success of females was assessed by the nonparametric χ2 test of independence. Mean hatching success, embryonic development, carapace length and width, weight, vitality test, humidity, and temperature were compared using the Student t test. Eight nests (4 from each treatment) were excluded from the analyses because they were inundated by high tide. All statistical analyses were performed using R software version 4.2.2 (R Core Team 2022) with a significance level of 5%.
RESULTS
Intrabeach Substrate Analysis. — The proportion of coarse sand differed significantly between the intrabeach study areas (CLN = 58.1% ± 6.6% SD, FDS = 29.4% ± 3.4% SD; t = 15.44, df = 30, p < 0.001; Fig. 2B). According to the Munsell colour charts, the northern part of the beach was lighter (CLN = 5Y 6/1, FDS = 2.5Y 8/4).
Nesting Success. — Over the 80 nights of the study, we registered 1086 nesting activities, unevenly distributed among the intrabeach zones (CLN = 44%, n = 478; FDS = 56%, n = 608; χ2 = 13.22, df = 1, p < 0.001). However, nesting success was not significantly different among the intrabeach zones (CLN = 49%, FDS = 55%; χ2 = 0.35, df = 1, p > 0.05).
Experimental Study. — The mean sand moisture in the experimental treatments was not significantly different, neither at the beginning of incubation (CLN = 3.3% ± 1.3% SD, FDS = 3.9% ± 1.3% SD; t = 1.42, df = = 30, p > 0.05) nor at the end (CLN = 3.6% ± 0.9% SD, FDS = 3.4% ± 0.63% SD; t = 0.42, df = 30, p > 0.05). The mean sand temperature had a difference of 0.1°C between the 2 studied areas of the beach (CLN = 30.1°C ± 1.2°C SD, FDS = 30.0°C ± 1.2°C SD; t = 4.18, df = 4566, p < 0.001).
Embryonic development success was significantly different between substrates (CLN = 60.8% ± 16.6% SD, FDS = 43.7% ± 20.8% SD; t = 2.23, df = 22, p < 0.05; Fig. 3A). Hatching success was significantly higher in the lighter sand from the northern part of the beach (CLN = 56.6% ± 15.8% SD, FDS = 40.1% ± 19.1% SD, t = 2.30, df = 22, p < 0.05; Fig. 3B).
Citation: Chelonian Conservation and Biology: Celebrating 25 Years as the World’s Turtle and Tortoise Journal 22, 2; 10.2744/CCB-1581.1
Hatchling Body Condition. — Hatchling biometrics (straight carapace length [SCL]) and agility (self-righting time) were significantly different as a function of sand treatment (SCL: CLN = 42.0 ± 1.7 mm SD, FDS = 40.3 ± 2.3 mm SD; t = 2.14, df = 22, p < 0.05; agility: CLN = 25.3 ± 19.9 sec SD, FDS = 60.6 ± 45.5 sec SD; t = 2.46, df = 15.06, p < 0.05; Figs. 3C, 3D). Carapace width and weight of experimental hatchlings were similar among treatments (width: CLN = 31.1 ± 1.5 mm SD, FDS = 30.0 ± 2.3 mm SD; t = 1.42, df = 22, p > 0.05; weight: CLN = 16.1 ± 1.6 g SD, FDS = 14.8 ± 1.9 g SD; t = 1.75, df = 22, p > 0.05).
DISCUSSION
The effect of intrabeach variation in substrate type on reproductive traits of sea turtles is still relatively unexplored, although it may affect functionally important traits of offspring, such as size and vitality. The results found here for loggerheads demonstrate variability in embryonic development, hatching success, and hatchling phenotype as a function of intrabeach variation in the incubation substrate, all of which are indicative parameters of reproductive success (see also Patino-Martinez et al. 2022b).
Correlations between incubation substrate types and hatchling phenotypes have been documented in recent years with interspecific (Saito et al. 2019; Suhaimi et al. 2020) and interbeach (Fadini et al. 2011; Tacchi et al. 2019) analysis, either in situ or under experimental conditions (Patino-Martinez et al. 2022b). These results provide one of the few examples to date of intrabeach correlation between substrate type and reproductive success.
Some of the phenotypic traits (carapace length and agility) showed significant variation between substrates on the same beach. Therefore, the intrabeach observations of substrate phenotype of hatchlings found here could differentially affect offspring survival as a function of hatchling traits (bigger and more agile is better), as has been found in previous studies (McGehee 1990; Gyuris 1994; Reece et al. 2002; Patrício et al. 2018; Martins et al. 2020), reflecting further variation in reproductive success.
The evidence obtained from these results supports the notion that the observed associations between the substrate and traits may be attributed to epigenetic variations that directly influence the control of phenotypic traits, including carapace size and hatchling vitality. In future studies, multifactorial analysis could be conducted to explore various factors linked to the substrate, including compactness and ease of hatchling emergence (Patiño-Martínez et al. 2010), particle size and origin (Patiño-Martínez et al. 2022b), chemical composition (e.g., calcium carbonate concentration; Simkiss 1962; Bustard and Greenham 1968), gas diffusion around the nest (Ackerman 1980; Garrett et al. 2010; Cheng et al. 2015), and others such as temperature and humidity (Booth 2017; Saito et al. 2019; Martins et al. 2020). Cross-incubation experiments can provide valuable insights into the specific effects of the incubation substrate on epigenetic processes and phenotypic variation.
Nesting females were found to exhibit similar nesting success rates, which indicate the number of nesting attempts on the beach that result in the successful laying of a nest, between the studied areas. Substrate type apparently did not determine the selection or rejection of the nesting site selected by the females. Indeed, there was no consistency between parity in nesting success and disparity in hatching success between areas. It is likely that other physical and biological factors, such as beach topography, vegetation cover, and accumulated beach organic material, have a greater impact on the nesting site selection compared to sand characteristics (Garmestani et al. 2000; Wood and Bjorndal 2000; Serafini et al. 2009; Cuevas et al. 2010; Trindade 2012; Patino-Martinez et al. 2017; Patrício et al. 2018).
The transgenerational effects of environmentally induced offspring traits suggest some intertwined ecological and evolutionary questions that have not been explored so far in sea turtles (Lockley et al. 2020). Here we highlight the possibility that the same maternal individual may produce offspring with different phenotypes, depending on the type of incubation sand, even within the same beach, opening new questions for specific studies of survival and adaptation in sea turtles (Salinas and Munch 2011).
Influences on progeny traits are important for population recruitment (e.g., size and escape velocity) and may act to enhance survival in heterogeneous and/or environmentally unpredictable habitats through mechanisms of the bet-edging strategy (Patino-Martinez et al. 2022a). The specific effects of choosing different nesting sites will generally act to spread the probability of selective pressures on embryos (Patino-Martinez et al. 2022a). Variation in reproductive success was due to differences between substrate types within the beach rather than the location of nests within the beach. A theoretical estimate of hatchling productivity (the number of hatchlings that would reach the sea with each substrate type; Patino-Martínez et al. 2021), using the mean hatching success from this study and the number of nests on the study beach in the period from 2018 to 2022, would indicate a difference of up to 43,800 more hatchlings in clutches incubated under the most favorable substrate conditions. Therefore, the importance of determining sands with the highest hatching success, even within the same beach, should be greatest in colonies or populations highly threatened with extinction, where the hatching success of each nest becomes of utmost importance.
(A) Map of Cabo Verde’s location on the West African coast and Cabo Verde archipelago. (B) The island of Maio. (C) Research area (Praiona Beach) with 2 study zones: coarse, light sand, northern (CLN) and fine, dark sand, southern (FDS).
(A) Intrabeach study blocks: Each block of 2 treatments (white = coarse and light sand from the northern zone [CLN] and black = fine and dark sand from the southern zone [FDS]) was installed during the same night of nest laying (different nests identified by numbers). (B) Proportion of sand grain size fractions of each zone.
Boxplots showing (A) the median of embryonic development success, (B) hatching success, (C) hatchling carapace length, and (D) self-righting time among intrabeach sand type (coarse, light sand, northern [CLN] and fine, dark sand, southern [FDS]). The box shows the interquartile ranges, the horizontal line shows the median value, and the whiskers show the range.
Contributor Notes
Handling Editor: Jeffrey A. Seminoff
