Metadata

Description
Stable Isotope Analysis
Dolphin skin samples were removed from the blubber layer using a methanol-rinsed stainless-steel blade by cutting as close to the interface between the two layers as possible without retaining any blubber. Dolphin samples (both skin and blubber) and prey samples (muscle tissue) were dried in an oven at 60 °C for 24-48 hr and then homogenized into a fine powder (DeNiro and Epstein, 1978). Lipids were removed only from dolphin samples using a 2:1 chloroform:methanol mixture for one minute with a solvent volume three times greater than the sample, left undisturbed for at least 45 min, centrifuged for 10 min, and then the supernatant was removed. After repeating this process three times, each dolphin sample was rinsed in deionized water and dried. The dried prey samples and lipid-extracted dolphin samples were then weighed, (0.4–0.7 mg) , and placed in a 4x6 mm tin capsule for SIA using a ThermoFinnigan Delta V isotope ratio mass spectrometer (IRMS) coupled with a NA 1500 NE elemental analyzer. Isotopic ratios are reported in the standard delta (δ) notation relative to the international standards of Vienna Pee Dee belemnite (δ13C) and atmospheric nitrogen (δ15N) using the following equation (1):
(1) δ(‰) = [(RSample/RStandard) – 1] x 1000
All stable isotope analyses (SIA) were completed at the Southeast Environmental Research Center Stable Isotope Laboratory at Florida International University. Analytical reproducibility was based on replicates of internal standards including bovine liver and glycine; variation among standards was 0.12‰ and 0.10‰ for δ13C and δ15N, respectively. The mean skin and blubber C: N ratios were less than 3, indicating an adequate lipid extraction (Lesage et al., 2010).

References:
DeNiro, M. J., & Epstein, S. (1978). Influence of diet on the distribution of carbon isotopes in animals. Geochimica et cosmochimica acta, 42(5), 495-506.

Lesage, A., Lelli, M., Gajan, D., Caporini, M. A., Vitzthum, V., Miéville, P., ... & Emsley, L. (2010). Surface enhanced NMR spectroscopy by dynamic nuclear polarization. Journal of the American Chemical Society, 132(44), 15459-15461.

Description
DNA extraction and sex identification
In order to sex sampled individual dolphins, DNA was extracted from skin tissue (< 2 mm) using a modified phenol/chloroform protocol from Sambrook et al. (1989) and described in detail in Beal et al. (2019). Concentrations of extracted DNA were checked by nanodrop and quality checked by gel electrophoresis. Aliquots at 30 ng/µL concentration were created for sex and age analysis procedures. Sex was identified by PCR and gel electrophoresis using the following PCR protocol: 10 µL H2O, 0.2 µL Thermo ScientificTM DreamTaqTM (5 U/ µL), 4 µL DreamTaq Polymerase Buffer, 1 µL dNTPs (2mM), 1 µL MgCl2 (25mM), 1 µL of Forward and Reverse ZFX primers (1 µM) and Forward SRY primer, and 0.5 µL Reverse SRY primer (1 µM) to which 1.5 µL of aliquoted DNA was added for a total volume of 21.2 µL for each sample. Thermocycler settings were as follows: initial denaturation of 95°C for 30s, followed by 30 cycles of 94°C for 30s, 51°C for 45s, and 72°C for 45s, then a final extension at 68°C for 5 min, ending with a final hold at 4°C. PCR products were run on a 2.5% agarose gel with Smart Glow pre-stain at 90 Voltz for approximately 1.5 hours and viewed with UV light.

References:
Beal, A. P., Kiszka, J. J., Wells, R. S., & Eirin-Lopez, J. M. (2019). The bottlenose dolphin epigenetic aging tool (BEAT): A molecular age estimation tool for small cetaceans. Frontiers in Marine Science, 6, 561.

Sambrook, J., Fritsch, E. F., & Maniatis, T. (1989). Molecular cloning: a laboratory manual (No. Ed. 2). Cold spring harbor laboratory press.

Description
Age was estimated for all individuals using the Bottlenose Epigenetic Aging Tool (BEAT; Beal et al. 2019). Extracted DNA was divided into 600 ng aliquots (20ng/µL) and subjected to bisulfite transformation and pyrosequencing using the established protocol for the BEAT (EpigenDx, Hopkinton, MA, USA). The % DNA methylation was used in the BEAT model equation (2) to estimate age for all individuals. The BEAT model was determined to have a root mean square error of 5.14 when cross validated using the leave one out cross validation (LOOCV) method. This means that individual age estimates produced by the BEAT model are within this error range (Beal et al., 2019). Using this potential error, both high and low age estimates were also used in analyses. High age estimates were used for this study.
(2) Age estimate= 8.602 + 4.392 (%DNA GRIA_5) – 1.149 (%DNA TET2_2)

References:
Beal, A. P., Kiszka, J. J., Wells, R. S., & Eirin-Lopez, J. M. (2019). The bottlenose dolphin epigenetic aging tool (BEAT): A molecular age estimation tool for small cetaceans. Frontiers in Marine Science, 6, 561.