Mother- Hatchling Isotopic Relationship in Green Turtle: Isotopic Niche-based Modelling

© 2024 Academia Sinica, Taiwan Open Access MotherHatchling Isotopic Relationship in Green Turtle: Isotopic Niche-based Modelling Şükran Yalçın Özdilek1, Bektaş Sönmez2,*, and Burcu Mestav3 1Çanakkale Onsekiz Mart University, Faculty of Arts and Sciences, Department of Biology, Çanakkale, Türkiye. E-mail: [email protected] (Özdilek) 2Sivas Cumhuriyet University, Suşehri Timur Karabal Vocational School, 58600, Suşehri, Sivas, Türkiye. *Correspondence: E-mail: [email protected] (Özdilek) 3Çanakkale Onsekiz Mart University, Faculty of Arts and Sciences, Department of Statistics, Çanakkale, Türkiye. E-mail: [email protected] (Mestav) Received 27 December 2023 / Accepted 26 June 2024 / Published 11 November 2024 Communicated by Chien-Hsiang Lin The isotopic discrimination between a mother and her hatchlings has been modelled in various vertebrates, including sea turtles. In addition to the linear relation between δ13C and δ15N isotope values of the mother – hatchling couple, there is missing data on the combined effects of both isotopes, which represent ecological niches of a species. The stable δ13C and δ15N isotope signatures of live hatchlings and their mother’s epidermis tissues were used in green turtles. The samples were taken from three main breeding beaches, Akyatan, Sugözü and Samandağ in Türkiye during the 2020 nesting season. δ15N and δ13C values of hatchlings were not significantly different from those of mothers. Significant relationships were found between hatchlings and their mothers in terms of δ13C value. Furthermore, when the hatchling isotopic niche size is known, the condition probabilities of estimating the mother’s isotopic niche size were 85.16% and 92.88% with the 0.95 and 0.99 alpha levels. In addition to showing a linear relationship between hatchlings and their mother’s single isotopic composition, the current study offers a novel insight that proposes a niche overlap concept using two isotopes to comprehend the mother-hatchling relationship of green turtles living in the eastern Mediterranean. Key words: Chelonia mydas, Stable isotopes, Hatchling isotope, Feeding ecology, Mediterranean BACKGROUND Sea turtles are lecithotrophic reptiles, meaning that embryos get their nutrients from egg yolk. The yolk formation process is known as Vitellogenesis, and during this process the yolk precursor proteins (vitellogenin; VTG and very low-density lipoprotein; VLDL) are produced in the maternal liver and the yolk proteins represent the proteins found in female blood (Van Dyke and Griffith 2018). Mothers transport yolk precursors to the ovarian follicles, and after fertilization and ovulation, the developing embryo consumes yolk. Transportation of the yolk into follicles begins storing 7–8 months before the breeding migration in sea turtles (Wibbels et al. 1990), and the nutrients in the egg are derived from the food consumed and stored by the female at that time (Kwan 1994; Hamann et al. 2003). The yolk is expected to have particular δ13C and δ15N values, reflecting the diet of the female before migrating to the breeding beach. It is expected that the isotopic values of the mother and the hatchling will have a high correlation, due to the hatchling tissue components being derived directly from the food sources consumed by the mother (Pilgrim 2007). The isotopic discrimination factor between the mother and the hatchlings (∆ = δmother – δhatchling) has been demonstrated previously in various vertebrates such as snakes (Pilgrim 2007), sharks (Vaudo et al. 2010), and birds (Hahn et al. 2012). This topic has also been studied in Caretta caretta (Zbinden et al. 2011; Citation: Özdilek SY, Sönmez B, Mestav B. 2024. MotherHatchling isotopic relationship in green turtle: isotopic niche-based modelling. Zool Stud 63:31. doi:10.6620/ZS.2024.63-31. Zoological Studies 63:31 (2024) doi:10.6620/ZS.2024.63-31 1 © 2024 Academia Sinica, Taiwan Frankel et al. 2012; Carpentier et al. 2015), Dermochelys coriacea (Caut et al. 2008) and Chelonia mydas (Chabot et al. 2019). Previous research used epidermis, keratin, or blood samples from mothers, together with egg yolk, dead or newly dead hatchlings from nests (Caut et al. 2008; Zbinden et al. 2011; Frankel et al. 2012; Carpentier et al. 2015; Chabot et al. 2019). A significant relationship between C. mydas maternal epidermis and unhatched eggs was recorded (Chabot et al. 2019). The association between the isotopic ratios of hatchling (yolk, unhatched, freshly dead, and dead) and mothers’ tissue is described by allocating just a single isotopic ratio, most often δ13C and δ15N (Chabot et al. 2019). However, based on Hutchison’s n-dimensional niche concept, the chemical structure of an animal is directly impacted by both bionomic and scenopoetic (δ15N and δ13C) ecological information (Newsome et al 2007). In addition to a linear relationship between single isotope values of the mother and the hatchling, the combined effects of both isotopes (δ13C and δ15N), which represent ecological niches of a species, may reflect the compatibility of niche spaces of the mother and hatchling. The niche region and niche overlap metric are used to find niche overlaps among ontogenetic or spatial subpopulations in the multidimensional ecological niches in various species (Swanson et al. 2015; Shipley et al. 2019; Gauffier et al. 2020). On the other hand, the formulation of niche overlaps between the mothers and their hatchlings may be another tool for understanding the isotopic discrimination between mothers and their offspring. It is expected that nesting females’ isotopic niches may overlap the niches of hatchlings. This finding will be valuable in fieldwork because, in contrast to the sampling of females, the sampling of hatchlings offers the potential for a larger sample size and a more straightforward fieldwork design. Thus, hatchlings may help to better understanding of the feeding ecology of green turtles. In order to study the feeding ecology of sea turtles, individuals must be sampled either in their feeding areas or on the nesting beach. The process of turtle sampling can be both difficult and expensive, especially in feeding areas. In addition, on nesting beaches, it can sometimes be difficult to sample sufficiently without disturbing the individual in a short time. In this case, the isotope values of the hatchling can be used, assuming that they represent the mother. However, the isotopic composition relationship between the hatchling and the mother needs to be demonstrated with high-confidence tests. This has been demonstrated in C. carreta and D. coriacea, but this metric is lacking for the Mediterranean population of green sea turtles. The estimation of stable isotope conversion formulae between mothers and their hatchlings is a crucial concern in the field of conservation research. This is particularly relevant for understanding the trophic ecology of sea turtles using the analysis of their hatchlings (Kaufman et al. 2014; Carpentier et al. 2015). In order to understand the feeding ecology of the Mediterranean population of green sea turtles, it is important to use particular techniques that are easy to sample, practical, and non-harmful. This study addressed this important issue and set two main objectives. One of the aims is to test the hypothesis that the isotope compositions of the mother and the hatchling will be similar in green turtles. If so, it will be possible to obtain green turtle specific conversion equations that can predict the isotopic value of the mother based on the regression between hatchling and mother tissue. The second aim of the study is to test the degree of niche overlap between the mother and the hatchling, assuming that the isotopic niche of hatchlings represents that of the mother’s. If the dual-isotope values of the hatchling represent their mother’s isotopic values, they represent the mother’s isotopic niche. MATERIALS AND METHODS Study area and sample collection This study was conducted at the three nesting beaches (Samandağ, Akyatan, Sugözü) located on the Eastern Mediterranean coast of Turkey during the 2020 nesting season (Fig. 1). Samandağ, Akyatan and Sugözü nesting beaches are the most prominent breeding areas of the entire Mediterranean (Casale et al. 2018). Therefore, a total of 19 nesting females from three beaches were used, assuming that they represented the Mediterranean (n = 8 for Samandağ, n = 8 for Akyatan, and n = 3 for Sugözü). Hatchling samples were gathered by randomly choosing 5 samples from each nest constructed by the mother turtles from whom the isotope samples were obtained. Before starting the sample collection, ethical approvals were obtained from the local ethical board for animal experiments (28/02/2018-1800028086). Sampling was carried out during the night patrol in the 2020 nesting season. Five people patrolled the beach at night to observe female nesting turtles. The turtles were tagged and measured after they laid their eggs. Metal tags were used and placed on the trailing edge of the left forelimb flipper, as recommended by Balazs (1999). After females nested, epidermal tissue samples were extracted by taking a piece 4–6 mm wide and 3 mm deep from the trailing edge of a fore flipper using a 5 mm disposable biopsy punch (Vanderklift et al. 2020). The nests of each female were marked with sticks page 2 of 8Zoological Studies 63:31 (2024) © 2024 Academia Sinica, Taiwan and protected with a cage to prevent predation. After nests hatched, 5 live hatchlings per nest were sampled using a 2 mm biopsy punch on the outermost projection of the marginal scute (Bjorndal et al. 2010; Türkozan et al. 2019). Hatchlings were examined in order to eliminate the decomposition effect of dead hatchlings, which was likely to significantly affect stable isotope values and lead to larger confidence intervals in discrimination factors (Frankel et al. 2012). Both adult females and hatchlings were released on the beach after the procedure was completed. All the tissues obtained from both adults and hatchlings were dried in an air circulating oven at 60°C for 24 hours, and these tissues were stored in vial tubes at -20°C in a deep freezer. Stable isotope analysis The tissue samples taken out of the deep freezer were kept in an oven (60°C) for one day, and then ground with a micro-dismemberator (1800 rpm/2 minutes) into powder. The powdered samples were weighed with a 1 ± 0.005 mg microbalance (Sartorius) and packed in 9 × 9 mm tin cups. Later, they were analysed by a continuous-flow isotope-ratio mass spectrometer (Thermo-Electron Flash EA 2000 elemental analyser (EA) coupled via a Thermo Finnigan ConFlo IV Interface to a Delta V Thermo Finnigan Isotope Ratio Mass Spectrometer). In the mass spectrophotometer, CH-6 Sucrose (Vienna Pee Dee Belemnite sample for 13C/12C (δ13CVPDB − 10.45‰ ± 0.04‰) and USGS-34 Potassium Nitrate (δ15Nair − 1.8‰ ± 0.2‰) standards were used for the carbon and nitrogen isotope ratios, respectively. The isotope ratios were calculated using the ratios in the tissue samples and the standard measured in the mass spectrophotometer as follows (Peterson and Fry 1987); X = [(RSample/ RStandard)-1] × 1000 where RSample: 15N/14N or 13C/12C ratio corresponding to the sample, RStandard: atmospheric nitrogen for 15N/14N or Vienna Pee Dee Belemnite sample for 13C/12C. Fig. 1. The sampling beaches in the Eastern Mediterranean (M and H indicate the number of mothers and hatchlings sampled, respectively). N page 3 of 8Zoological Studies 63:31 (2024) © 2024 Academia Sinica, Taiwan Data Analysis In order to estimate the relationship between the isotopic composition of the mother (a dependent variable) and their hatchlings (an independent variable), linear regression was performed. In order to estimate the unknown parameters, a linear regression model was used. This method minimizes the sum of the squares in the difference between the observed and predicted values of the dependent variable configured as a straight line. Y = βo + βX + ϵ In this formula, Y represents the mother’s δ13C and δ15N values and X represents δ13C and δ15N values of the hatchlings. The average values of δ13C and δ15N for five hatchlings from each clutch, which are representative of the mothers, were considered. This model was used for all beaches combined. We calculated the isotopic niche area and niche overlap metrics between mothers and hatchlings using the R package nicheROVER v1.0 (Swanson et al. 2015). The overlap ( ) function was used with δ13C and δ15N values. Ellipses were fitted to both the mother and the hatchlings, and the mean overlap was calculated using Bayesian 95% credible intervals based on 10000 simulations. Because the overlap metric is directional, the mean overlap represents the probability that an individual from a hatchling will be found in the niche of its mother. This method does not assume a homogeneous distribution of individuals inside the niche region and is applicable to small and uneven sample sizes (Swanson et al. 2015). RESULTS Descriptive statistics of isotope values of epidermis tissue in nesting beaches according to hatchlings and their mothers are shown in table 1. Although the hatchlings have a lower δ13C value compared to their respective mothers, this difference is not statistically significant (Paired t-test = 0.30, d.f. = 18, p > 0.05). Also, the δ15N value of the hatchlings was not significantly higher than the δ15N values of their mothers (Paired t-test = 2.09, d.f. = 18, p = 0.05). The models for δ13C isotope value between hatchling and mother epidermis samples overall showed significant relationships between them (Fig. 2). However, there is no statistically significant relationship between the δ15N isotope value of hatchlings and that of the mother’s epidermis. The conversion model equations, which indicate a significant relationship between hatchling and female epidermis, are presented in table 1. The niche plots, density distributions, and raw data for each pairwise combination of isotope data for both mothers and hatchlings (i.e., bivariate projections of 3-dimensional isotope data) are shown in figure 3. The niche sizes of mothers and hatchlings were estimated as 26.5‰ ± 5.96‰ and 20.3‰ ± 5.16‰, respectively. The hatchlings were found in the niche of mothers in 85.2% and 92.8% of α = 95 and α = 99 levels, respectively. DISCUSSION This study is the first to report the isotopic relationship and conversion equations between hatchling and mother green turtles of the eastern Mediterranean. We determined that the δ15N and δ13C values of hatchlings did not exhibit any significant differences compared to those of mothers. Literature review shows that the δ13C value of the unhatched egg was found to be depleted and the δ15N value to be higher in green turtles (Chabot et al. 2019). Similarly, freshly dead hatchlings in loggerhead turtles have been reported to have higher δ15N and depleted δ13C values compared to their respective mothers (Frankel et al. 2012). Our results showed that δ15N values were slightly enriched compared to their mothers, similar to previous studies. Table 1. The descriptive statistics of isotope values of epidermis tissue in eastern Mediterranean green turtles according to hatchlings and nesting females and the conversion model equations between them (the isotope values of the hatchlings in each clutch were calculated based on the intra-clutch mean of the hatchling isotope values) nMean ± Sd (‰) Min-max (‰) Regression equation and “p” values Hatchling δ13C 95 -9.24 ± 1.93 -12.12 – -7.17 δ13Cmother = -5.48 + 0.395*δ13Chatchling R2 = 0.29 p < 0.001 Mother 19 -9.12 ± 1.42 -11.82 – -7.36 Hatchling δ15N 95 6.41 ± 0.69 5.03 – 7.59 δ15Nmother = 3.62 + 0.342* δ15Nhatchling R2 = 0.04 p > 0.05 Mother 19 5.82 ± 1.17 4.34 – 8.12 page 4 of 8Zoological Studies 63:31 (2024) © 2024 Academia Sinica, Taiwan The higher values of δ15N in hatchlings is consistent with the idea that the hatchlings’ trophic level is higher than their mothers’. Since hatchlings consume egg yolk produced by their mother, they are expected to have a higher trophic level (Partridge et al. 2001; Carpentier et al. 2015). A positive relationship was found between the isotope values of mothers and their hatchlings. This suggests that stable isotope analysis of hatchlings is a viable method to assess foraging ecological questions in sea turtles. Therefore, the sampling from hatchlings can be used as a less invasive procedure to understand the diet of adult sea turtles instead of techniques such as gastric lavage or blood sampling during nesting. Similar relationships have been found and proposed between various tissues of sea turtles and their hatchlings and/ or eggs. However, model fit R2 values in the present study are lower than in other similar studies between the mother and the hatchling in regression conversion equations. For instance, R2 values in green turtles from the epidermis to unhatched egg conversion equations in the δ13C and δ15N models are R2 = 0.70 and R2 = 0.64 respectively (Chabot et al. 2019). Carpentier et al. (2015) reported R2 = 0.83 and R2 = 0.86 for δ13C and δ15N models, respectively, in loggerhead turtles. Similarly, Caut et al. (2008) obtained conversion equations between egg-yolk and plasma and red blood cells in leatherback sea turtles. They reported that R2 values for Fig. 2. The relationships of the mother and hatchling δ13C and δ15N epidermis values for eastern Mediterranean green turtles with 95% confidence (grey) and predicted intervals (red), and conversion model equations (The red boxes indicate the mother and hatchling box plots. In the box plots, the medians of the mother and hatchling δ13C values are -8.85‰ and -8.177‰, respectively, and the medians of the mother and hatchling δ15N values are 5.38‰ and 6.52‰, respectively. The 25%–75% interquartile range of the distribution is -9.76‰ and -7.92‰ for the δ13C values of the mother, and -11.48‰ and -7.8‰ for the hatchling. The 25%–75% interquartile range of the distribution for δ15N values is 4.82‰ and 6.60‰ for the mother and 5.97‰ and 6.96‰ for the hatchling. The minimum and maximum values are given in Table 1). page 5 of 8Zoological Studies 63:31 (2024) © 2024 Academia Sinica, Taiwan δ13C and δ15N models were 0.84 and 0.89 respectively, between egg yolk and plasma, and 0.86 and 0.84 between egg yolk and red blood cell. The lower explanatory power (R2) of our study for the δ13C and δ15N models may be due to the possibility that the relationship between a mother’s and her hatchlings’ epidermides for isotope discrimination factors is weaker in green turtles than in other sea turtle species. A similar situation was reported by Chabot et al. (2019) for lower explanatory power between unhatched egg contents and female epidermis in green turtles. Frankel et al. (2012) found an r value for δ13C (r = 0.24) lower than our study between fresh-dead hatchlings and their mothers in loggerhead turtles. Moreover, they noted that fresh-dead hatchlings showed less variation than the dead in the nest, and fresh-dead hatchlings would provide a more acceptable estimation. Hatchling tissue collection in this study involved easy sampling for isotope analysis on nesting beaches. Sufficient samples can easily be obtained from a 2-mm biopsy punch, which does not require killing the hatchlings. Similarly, Frankel et al. (2012) noted that hatchlings could be sampled using a 2 mm biopsy punch. This will not affect the growth rate and health status of hatchlings (Bjorndal et al. 2010). Based on the results of the study, hatchling tissues can indicate their mother’s foraging ecology. However, since we investigated a single maternal tissue (epidermis), this may be limited to reflecting the mother, considering that the tissues had different incorporation and turnover rates. Therefore, it is important to reveal the relationships and differences between other tissues such as blood (plasma and red blood cell) of the mother and the tissue of the hatchling in future studies of green sea turtles. A positive relationship was found between the δ13C isotope values of mothers and their hatchlings, but not between the δ15N values. The limited correlation between the isotopic nitrogen content of hatchlings and their mothers may be due to a lack of differentiation between hatchlings and mothers, rather than the small sample size. The observed difference in the discriminant values of nitrogen and carbon between hatchlings and their mothers, as well as the findings published in the literature (Carpentier et al. 2015; Frankel et al. 2012), suggest that there may be variability in the transfer of carbon and nitrogen sources from the mother to hatchlings. Therefore, the use of dual isotopes presents a more viable approach for comprehending the ecological functions of sea turtles. Isotopic niche analysis is a valuable tool for studying ecological niches due to the stable isotopes of sea turtles, which reflect both their prey and their Fig. 3. Each pairwise combination of isotope data for both mothers and hatchlings on green turtle nesting beaches, a), Carbon density distributions of mother and hatchling, b) Niche plot graph of mother and hatchling, c) Dot plot of carbon and nitrogen values of mother and hatchling (the isotope values of the hatchlings in each clutch were calculated based on the intra-clutch mean of the hatchling isotope values), d) Nitrogen density distributions of mother and hatchling. page 6 of 8Zoological Studies 63:31 (2024) © 2024 Academia Sinica, Taiwan environment. The generalist vs. specialist feeding strategy is particularly attributed to the wideness of isotopic niche space and the individual variation in resource use, which are taken into consideration to assess the feeding strategy of loggerhead turtles using stable isotopes (Vander Zanden et al. 2010). The large niche area of mothers is explained by the more generalist feeding strategy of green turtle populations. Even though the predictability of the mother’s isotopic ratio from the hatchlings was remarkably high, the variation in the mother’s epidermal samples still has to be taken into account. This indicates a possibility of the realization of food sources transferred by mothers to their hatchlings in the last few months with various foraging strategies. This finding is consistent with the polymorphic foraging strategy of each individual throughout their life span (Seminoff et al. 2007; Ferreira et al. 2021). CONCLUSIONS Stable isotope analysis provides valuable information about resource use in the trophic ecology of green turtles. The hatchlings reflect the isotopic composition of their mothers since they use the nutrients stored in the egg yolk during vitellogenesis. There was a significant relationship with a linear regression model between the mother and the hatchlings. In addition, there is a high probability that an individual hatchling was found in the niche area of its respective mother. However, the temporal and spatial intrapopulation variation in foraging strategies suggested that while assessing mother-hatchling isotopic compatibility, intrapopulation differences should also be considered. Acknowledgements: This study was supported by TUBITAK (Project code: 118Y431). We thank Selma Kırbeci, Mehmet Sert, Mehmet Tural, Ömür Özkan, Güven Aslan, and all the volunteers who worked in the survey. We would also like to express our gratitude to WWF-Turkey and, Samandağ Environmental Protection and Tourism Association, and 7th Regional Directorate of Nature Conservation and National Parks of the Republic of Turkey Ministry of Agriculture and Forestry. Funding: This study is financially supported by The Scientific and Technological Research Council of Turkey (TUBITAK) with project code 118Y431. Authors’ contributions: Şükran Yalçın Özdilek (SYO) conceived the ideas and designed methodology; Bektaş Sönmez (BS) and SYO collected the data; Burcu Mestav (BM) and SYO analysed the data; all authors wrote the manuscript and contributed critically to the drafts and gave final approval for publication. Competing interests: The authors have no relevant conflicts of interest to declare. Availability of data and materials: The data and analysis are transformed to R documents and data that support the findings of this study and are available from the authors, upon reasonable request. Consent for publication: Not applicable. Ethics approval consent to participate: All applicable international guidelines for sampling, care, and experimental use of organisms for the study were followed and all necessary approvals were obtained. REFERENCES Balazs GH. 1999. Factors to consider in the tagging of sea turtles. 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