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Inherent Variation in Stable Isotope Values and Discrimination
Factors in Two Life Stages of Green Turtles
* Corresponding author; e-mail: [email protected]
Physiological and Biochemical Zoology 85(5):431441. 2012. 2012 by TheUniversity of Chicago. All rights reserved. 1522-2152/2012/8505-1175$15.00.DOI: 10.1086/666902
Hannah B. Vander Zanden1,*Karen A. Bjorndal1
Jose Miguel Ponciano3
Alan B. Bolten11Archie Carr Center for Sea Turtle Research and Departmentof Biology, University of Florida, P.O. Box 118525,Gainesville, Florida 32611; 2Cayman Turtle Farm, P.O. Box812, Grand Cayman KY1-1301, Cayman Islands;3Department of Biology, University of Florida, P.O. Box118525, Gainesville, Florida 32611
Accepted 5/26/2012; Electronically Published 8/3/2012
Online enhancements: appendixes.
We examine inherent variation in carbon and nitrogen stableisotope values of multiple soft tissues from a population ofcaptive green turtles Chelonia mydas to determine the extentof isotopic variation due to individual differences in physiology.We compare the measured inherent variation in the captivepopulation with the isotopic variation observed in a wild pop-ulation of juvenile green turtles. Additionally, we measure diet-tissue discrimination factors to determine the offset that occursbetween isotope values of the food source and four green turtletissues. Tissue samples (epidermis, dermis, serum, and redblood cells) were collected from captive green turtles in twolife stages (40 large juveniles and 30 adults) at the CaymanTurtle Farm, Grand Cayman, and analyzed for carbon andnitrogen stable isotopes. Multivariate normal models were fitto the isotope data, and the Bayesian Information Criterionwas used for model selection. Inherent variation and discrim-ination factors differed among tissues and life stages. Inherentvariation was found to make up a small portion of the isotopicvariation measured in a wild population. Discrimination factorsnot only are tissue and life stage dependent but also appear tovary with diet and sea turtle species, thus highlighting the needfor appropriate discrimination factors in dietary reconstruc-tions and trophic-level estimations. Our measures of inherentvariation will also be informative in field studies employing
stable isotope analysis so that differences in diet or habitat aremore accurately identified.
Stable isotope analysis is commonly used to investigate con-sumer foraging patterns in ecological studies. Dietary recon-structions through mixing models and trophic-level estimationsrely on diet-tissue discrimination factors (the difference be-tween stable isotope values of an organisms tissue and diet).More recent applications using carbon and nitrogen stable iso-tope compositions (d13C and d15N, respectively) to examinetrophic niche and specialization rely on measures of stable iso-tope variance within the population (Araujo et al. 2007; Laymanet al. 2007b; Newsome et al. 2007; Vander Zanden et al. 2010).The isotopic niche is used as a proxy for ecological dimensionsof resource use because the stable isotope ratios in the tissueof an organism represent the assimilated diet (Layman et al.2007a; Vaudo and Heithaus 2011). Additionally, specializationcan be inferred by examining the isotopic variation of a pop-ulation or an individual through time. Low variation indicatesspecialization, while substantial variation indicates generaliza-tion (Bearhop et al. 2004; Martnez del Rio et al. 2009a; New-some et al. 2009; Vander Zanden et al. 2010).
In many studies, isotopic variation is attributed to diet andhabitat differences, but it can also result from variation in theisotopic composition within a prey species, inherent variationin the consumer, and measurement error (Bearhop et al. 2002;Matthews and Mazumder 2004; Phillips and Eldridge 2006;Barnes et al. 2008). Inherent variation in stable isotope values(hereafter referred to as inherent variation) is a consequenceof isotopic deviations that arise from individual differences inphysiology despite consuming the same diet and experiencingcontrolled conditions. Although not often quantified, inherentvariation could substantially affect conclusions based on stableisotope data. Inherent variation can depend on the species, life-history stage, and environment (Barnes et al. 2008), yet mea-surements of such variation from animals on controlled dietsare sparse (Matthews and Mazumder 2004; Sweeting et al. 2005;Barnes et al. 2008; Seminoff et al. 2009). In one case, inherentvariation made up a large portion of the isotopic variance mea-sured in a wild population of sea bass Dicentrachus labrax(Barnes et al. 2008). Therefore, inherent variation should beconsidered when generating inferences about foraging patternsin wild populations. If it is assumed that all isotopic variationobserved is due to differences in diet and habitat use, then the
432 H. B. Vander Zanden, K. A. Bjorndal, W. Mustin, J. M. Ponciano, and A. B. Bolten
resulting isotopic niche or level of generalization may beoverestimated.
Diet-tissue discrimination is represented as D p d tissueand results from processes such as fractionation duringddiet
metabolic transformations and isotopic routing (Martnez delRio et al. 2009b). Accurate diet-tissue discrimination factorsare essential to estimate trophic level and reconstruct diets, andvariation in the discrimination factor should be accounted forin mixing models (Post 2002; Wolf et al. 2009). Many studieshave used generalized discrimination factors because of the lackof species-specific values, yet the use of such values can leadto large errors or meaningless results in the output of mixingmodels (Caut et al. 2009).
Consumer tissues are often enriched in 15N and 13C comparedwith their diets (DeNiro and Epstein 1978; DeNiro and Epstein1981; Post 2002), though discrimination factors may vary withlife stage, environment, form of nitrogenous waste excretion,taxon, species, tissue, diet quality, and diet isotopic composition(Vander Zanden and Rasmussen 2001; Vanderklift and Ponsard2003; Caut et al. 2009). The commonly used diet-tissue dis-crimination value for nitrogen (D15N) is 3.4 (DeNiro andEpstein 1981; Post 2002). Values of D13C are typically muchsmaller than D15N values, resulting in a reduced trophic shiftin d13C values as nutrients are transferred through the foodweb (DeNiro and Epstein 1978).
The first objective of our study was to quantify the inherentvariation in a captive population of green turtles Chelonia my-das fed a consistent diet. We examined the variation in stableisotope valuesour measure of inherent variationin fourtissue types (epidermis, dermis, serum, and red blood cells)and two life stages (large juveniles and adults). We then com-pared our measure of inherent variation in epidermis with theisotopic variance observed in a wild population. The secondobjective of our study was to measure discrimination factorsfor each of the four tissues in both juvenile and adult greenturtles maintained on an isotopically consistent diet. Further-more, we incorporated the measure of inherent variation inour estimates of the discrimination factors. We also comparedthe discrimination factors found here with other sea turtlespecies.
Material and Methods
Green turtles were housed at the Cayman Turtle Farm (CTF)in Grand Cayman, British West Indies. These turtles are de-scendants of a mixed breeding stock made up of turtles fromat least four nesting populations (Wood and Wood 1980).Adults ranged from 10 to approximately 70 yr of age, from 92to 110 cm curved carapace length (CCL), and from 75 to 186kg. The large juveniles were approximately 46 yr of age andhad been raised in captivity. Their size ranged from 64 to 92cm CCL and 30 to 63 kg. At CTF, large juveniles grow atsubstantially higher rates (about 14 cm CCL/yr) than the samesize class in the wild (Wood and Wood 1993; Bjorndal et al.
2000), and adults at CTF grow very little, if at all, after sexualmaturity (Wood and Wood 1993).
The turtles were fed an extruded floating pellet diet man-ufactured by Southfresh Feeds at 0.5% body weight/d for 4 yrbefore sampling. The feed consists of at least 36% crude protein,3.5% crude fat, 12% moisture, 6% crude fiber, and 1% phos-phorus. A complete list of the feed ingredients is available inappendix A in the online edition of Physiological and Biochem-ical Zoology. The diet is highly digestible, and a similar diet(35% protein and 3.9% fat) had a dry-matter digestibility of85.9% and a protein digestibility of 89.4% (Wood and Wood1981). The turtles were assumed to be at isotopic equilibriumwith the diet.
Juveniles and adults were maintained in tanks or an artificialpond. The water intake pipes for each were directed to createa slow circular current against which the turtles swam. Theywere almost constantly in motion during daylight hours, withresting periods at night. The maximum depth of the adult pondwas 5.2 m, and there was an artificial beach available for femalesto lay eggs. The depth of the juvenile tanks was 0.9 m.
During April and May 2010, tissue samples were collected from30 adult female green turtles and from 40 large juvenile greenturtles. Blood samples of 28 mL were drawn from the carotidarteries using sterile 16G # 2 IV catheters (SURFLO I.V. cath-eters) and were immediately transferred to 9-mL drawCORVAC serum separator tubes. Serum and red blood cellswere separated by centrifugation at 2,195 g and frozen sepa-rately at 20C until analysis. Skin samples were taken with6-mm Miltex sterile biopsy punches in the region between thefront flipper and the head, just below the carapace, and placedin 70% ethanol. Isotope values of sea turtle epidermis preservedin ethanol were