stable isotopic comparison between loggerhead sea turtle tissues

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Stable isotopic comparison between loggerhead sea turtle tissues Hannah B. Vander Zanden 1,2 *, Anton D. Tucker 3 , Alan B. Bolten 2 , Kimberly J. Reich 4 and Karen A. Bjorndal 1 1 Archie Carr Center for Sea Turtle Research and Department of Biology, University of Florida, Gainesville, FL 32611, USA 2 Department of Geology and Geophysics, University of Utah, Salt Lake City, UT 84112, USA 3 Mote Marine Laboratory, Sarasota, FL 34236, USA 4 Department of Marine Biology, Texas A&M University, Galveston, TX 77553, USA RATIONALE: Stable isotope analysis has been used extensively to provide ecological information about diet and foraging location of many species. The difference in isotopic composition between animal tissue and its diet, or the diet-tissue discrimination factor, varies with tissue type. Therefore, direct comparisons between isotopic values of tissues are inaccurate without an appropriate conversion factor. We focus on the loggerhead sea turtle (Caretta caretta), for which a variety of tissues have been used to examine diet, habitat use, and migratory origin through stable isotope analysis. We calculated tissue-to-tissue conversions between two commonly sampled tissues. METHODS: Epidermis and scute (the keratin covering on the carapace) were sampled from 33 adult loggerheads nesting at two beaches in Florida (Casey Key and Canaveral National Seashore). Carbon and nitrogen stable isotope ratios were measured in the epidermis and the youngest portion of the scute tissue, which reect the isotopic composition of the diet and habitat over similar time periods of the order of several months. RESULTS: Signicant linear relationships were observed between the δ 13 C and δ 15 N values of these two tissues, indicating they can be converted reliably. CONCLUSIONS: Whereas both epidermis and scute samples are commonly sampled from nesting sea turtles to study trophic ecology and habitat use, the data from these studies have not been comparable without reliable tissue-to-tissue conversions. The equations provided here allow isotopic datasets using the two tissues to be combined in previously published and subsequent studies of sea turtle foraging ecology and migratory movement. In addition, we recommend that future isotopic comparisons between tissues of any organism utilize linear regressions to calculate tissue-to-tissue conversions. Copyright © 2014 John Wiley & Sons, Ltd. Stable isotope analysis has become a widely used tool in ecological studies. Carbon and nitrogen isotopes reect an integration of diet, habitat, and geographic location, and, therefore, these ecogeochemical tracers can provide information about foraging ecology and movement. [13] Additional logistical challenges arise in studying highly migratory marine organisms. Sea turtles have complex life cycles that span ontogenetically distinct and disparate habitats. As adults, they make regular migrations between foraging and reproductive areas, [4,5] making it difcult for researchers to follow individuals across seasons. Loggerhead sea turtles (Caretta caretta) occur throughout the temperate and tropical oceans globally [6] but are considered endangered under the United States Endangered Species Act of 1973. Threats such as incidental capture in sheries, human disturbance at nesting beaches, and climate-induced changes to water and nesting beach temperatures may prevent population recovery. [7,8] The use of stable isotope analysis has illuminated facets of sea turtle ecology that are otherwise difcult to study, such as the habitat used during cryptic juvenile life stages and foraging area origin of nesting females. [911] Studies of loggerhead ecology employing stable isotopes have increased over the last decade, with 11 tissue types used in studies to date (Table 1). However, the stable isotope values among these tissues are not directly comparable due to the processes that may differentially affect the isotopic composition of tissues as nutrients are integrated from the diet. Stable isotope values of carbon and nitrogen (δ 13 C and δ 15 N) are known to differ among different tissues within an individual as a result of multiple factors, including fractionation (the consequence of mass differences between isotopes that cause changes in isotopic abundance between reactants and products) and isotopic routing (the process of routing macronutrients such as proteins, fats, and carbohydrates directly from the diet to tissue without homogenization and resynthesis of molecules). [12] The isotopic offset between the composition of a dietary item and the animal tissues is referred to as the diet-tissue discrimination factor, and this difference is typically larger for δ 15 N values than for δ 13 C values. [2,1315] Not only is there variation in diet-tissue discrimination factors among tissues, but these discrimination factors can differ among individuals * Correspondence to: H. B. Vander Zanden, Department of Geology and Geophysics, University of Florida, 115 S 1460 E, Salt Lake City, UT 84112, USA. E-mail: [email protected] Copyright © 2014 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2014, 28, 20592064 Research Article Received: 15 May 2014 Revised: 21 July 2014 Accepted: 22 July 2014 Published online in Wiley Online Library Rapid Commun. Mass Spectrom. 2014, 28, 20592064 (wileyonlinelibrary.com) DOI: 10.1002/rcm.6995 2059

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Page 1: Stable isotopic comparison between loggerhead sea turtle tissues

Research Article

Received: 15 May 2014 Revised: 21 July 2014 Accepted: 22 July 2014 Published online in Wiley Online Library

Rapid Commun. Mass Spectrom. 2014, 28, 2059–2064

Stable isotopic comparison between loggerhead sea turtle tissues

Hannah B. Vander Zanden1,2*, Anton D. Tucker3, Alan B. Bolten2, Kimberly J. Reich4 andKaren A. Bjorndal11Archie Carr Center for Sea Turtle Research and Department of Biology, University of Florida, Gainesville, FL 32611, USA2Department of Geology and Geophysics, University of Utah, Salt Lake City, UT 84112, USA3Mote Marine Laboratory, Sarasota, FL 34236, USA4Department of Marine Biology, Texas A&M University, Galveston, TX 77553, USA

RATIONALE: Stable isotope analysis has been used extensively to provide ecological information about diet andforaging location of many species. The difference in isotopic composition between animal tissue and its diet, or thediet-tissue discrimination factor, varies with tissue type. Therefore, direct comparisons between isotopic values of tissuesare inaccurate without an appropriate conversion factor. We focus on the loggerhead sea turtle (Caretta caretta), for whicha variety of tissues have been used to examine diet, habitat use, and migratory origin through stable isotope analysis. Wecalculated tissue-to-tissue conversions between two commonly sampled tissues.METHODS: Epidermis and scute (the keratin covering on the carapace) were sampled from 33 adult loggerheads nestingat two beaches in Florida (Casey Key and Canaveral National Seashore). Carbon and nitrogen stable isotope ratios weremeasured in the epidermis and the youngest portion of the scute tissue, which reflect the isotopic composition of the dietand habitat over similar time periods of the order of several months.RESULTS: Significant linear relationships were observed between the δ13C and δ15N values of these two tissues, indicatingthey can be converted reliably.CONCLUSIONS: Whereas both epidermis and scute samples are commonly sampled from nesting sea turtles to studytrophic ecology and habitat use, the data from these studies have not been comparable without reliable tissue-to-tissueconversions. The equations provided here allow isotopic datasets using the two tissues to be combined in previouslypublished and subsequent studies of sea turtle foraging ecology and migratory movement. In addition, we recommendthat future isotopic comparisons between tissues of any organism utilize linear regressions to calculate tissue-to-tissueconversions. Copyright © 2014 John Wiley & Sons, Ltd.

(wileyonlinelibrary.com) DOI: 10.1002/rcm.6995

Stable isotope analysis has become a widely used tool inecological studies. Carbon and nitrogen isotopes reflect anintegration of diet, habitat, and geographic location, and,therefore, these ecogeochemical tracers can provideinformation about foraging ecology and movement.[1–3]

Additional logistical challenges arise in studying highlymigratory marine organisms. Sea turtles have complex lifecycles that span ontogenetically distinct and disparatehabitats. As adults, they make regular migrations betweenforaging and reproductive areas,[4,5] making it difficult forresearchers to follow individuals across seasons.Loggerhead sea turtles (Caretta caretta) occur throughout the

temperate and tropical oceans globally[6] but are consideredendangered under the United States Endangered Species Actof 1973. Threats such as incidental capture in fisheries, humandisturbance at nesting beaches, and climate-induced changesto water and nesting beach temperatures may preventpopulation recovery.[7,8] The use of stable isotope analysis has

* Correspondence to: H. B. Vander Zanden, Department ofGeology and Geophysics, University of Florida, 115 S1460 E, Salt Lake City, UT 84112, USA.E-mail: [email protected]

Rapid Commun. Mass Spectrom. 2014, 28, 2059–2064

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illuminated facets of sea turtle ecology that are otherwisedifficult to study, such as the habitat used during crypticjuvenile life stages and foraging area origin of nestingfemales.[9–11] Studies of loggerhead ecology employing stableisotopes have increased over the last decade, with 11 tissuetypes used in studies to date (Table 1). However, the stableisotope values among these tissues are not directly comparabledue to the processes that may differentially affect the isotopiccomposition of tissues as nutrients are integrated from the diet.

Stable isotope values of carbon and nitrogen (δ13C andδ15N) are known to differ among different tissues within anindividual as a result of multiple factors, includingfractionation (the consequence of mass differences betweenisotopes that cause changes in isotopic abundance betweenreactants and products) and isotopic routing (the process ofrouting macronutrients such as proteins, fats, andcarbohydrates directly from the diet to tissue withouthomogenization and resynthesis of molecules).[12] Theisotopic offset between the composition of a dietary itemand the animal tissues is referred to as the diet-tissuediscrimination factor, and this difference is typically largerfor δ15N values than for δ13C values.[2,13–15] Not only is therevariation in diet-tissue discrimination factors among tissues,but these discrimination factors can differ among individuals

Copyright © 2014 John Wiley & Sons, Ltd.

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Table 1. Studies that have used stable isotope analysis toexamine loggerhead ecology and life history divided bytissue type and life stage

Loggerhead tissue Life stage Source

Scute Adult [23,34,35]

Juvenile [36]

Epidermis Adult [11,28,37–40]

Juvenile [20,36,41–44]

Hatchling [20,38]

Whole blood Juvenile [36]

Red blood cells Adult [39,45]

Juvenile [20,46]

Hatchling [20]

Plasma Adult [39]

Juvenile [20,46,47]

Hatchling [20]

Muscle Juvenile [36,44]

Hatchling [48]

Bone Adult [49]

Juvenile [50–52]

Yolk Egg [9,35,53–55]

Whole egg Egg [50]

Whole embryo Egg [50]

Whole hatchling Hatchling [50]

H. B. Vander Zanden et al.

2060

as a consequence of other factors, including life stage,nutritional status, diet composition, and extent of dietaryrouting.[2,16–19] Tissues can also have different isotopicresidence times, depending on the rate of incorporation andprotein turnover.[2,12,20] Therefore, caution may be necessaryin comparing tissues that reflect different time periods if thediet is not consistent through time.[21,22] Loggerheads exhibitremarkable isotopic consistency through time, indicating thattheir resource use varies little over periods of up to 12 years.[23]

Thus, most loggerhead tissues should be comparable forindividuals of a similar life stage and nutritional status.As a result of tissue-specific discrimination factors, the

isotopic composition of distinct tissues cannot be comparedwithout appropriate conversion factors. We use the term’diet-tissue discrimination’ to refer only to the isotopicdifference that occurs between consumer tissues and theirdiets, consistent with the definition of Martínez del Rio andWolf.[12] The isotopic difference between tissues is not a’discrimination factor’, and therefore we use the term’tissue-to-tissue conversion’ to refer to the comparison ofdistinct tissue types. There have been few studies that providetissue-to-tissue conversions for loggerhead sea turtles.The goal of this study was to determine the tissue-to-tissue

conversions between adult loggerhead scute and epidermistissue stable isotope values. Scute is the continuouslygrowing but inert keratin-based tissue found on the carapaceof hard-shelled sea turtles.[24] Scute and epidermis samplescan be collected from live turtles without affecting the healthor physiological status of the individuals sampled,[25] butbone and muscle biopsies are much more invasive. Bloodoften requires immediate centrifugation and freezing, whichcan present logistical challenges for field collections whereelectricity is not readily available. Therefore, scute andepidermis are commonly used tissues in field collections, asthe samples can be stored at room temperature in ethanolwithout alteration to the isotope ratios.[26] The conversion

wileyonlinelibrary.com/journal/rcm Copyright © 2014 John Wile

equations that we provide here will permit the comparisonof these tissues in future and previously published datasets,and therefore further our understanding of the foragingecology and habitat use of loggerhead sea turtles.

EXPERIMENTAL

Field collections

Nesting female loggerheads were sampled at Cape CanaveralNational Seashore (CNS), on the east coast of Florida in 2004(n = 13), and Casey Key (CK), on the west coast of Florida, in2011 (n = 20) between the months of May and July. Prior tocollection, the sampling site was cleaned with isopropylalcohol. Scute and skin were collected with 5 or 6 mm biopsypunches. The skin samples contained both epidermal anddermal tissue, which were later separated. Scute sampleswere collected from the posterior medial region of the thirdright lateral scute and stored in 70% ethanol. At CK, skinsamples were taken from the trailing margin of a rear flipper,and, at CNS, skin samples were taken from the ’shoulder’region between the front flipper and the head, and stored in70% ethanol. Ethanol does not significantly alter the stableisotope values of loggerhead epidermis,[26] and thereforewas assumed not to alter the isotopic values of scute. Samplesfrom CNS were stored for up to 4 years and those from CKwere stored for up to 5 months prior to analysis. There havebeen no studies of the long-term storage effects on the stableisotope values of sea turtle tissues; however, there were nosignificant effects of 70% ethanol preservation on the δ13C orδ15N values of sea turtle epidermis from periods ranging from1 to 60 days.[26]

Sample preparation

In the lab, skin samples were rinsed with deionized water,and the surface epidermis was separated from the dermaltissue and processed further. CNS epidermis samples werehomogenized with a scalpel blade prior to drying at 60°Cfor a minimum of 24 h. The epidermis from CK was freeze-dried at –55°C for 8 h and ground to a small size (0.05 mmparticle diameter). Lipids were removed from epidermisusing an ASE300 accelerated solvent extractor (Dionex,Sunnyvale, CA, USA) and petroleum ether solvent for threeconsecutive cycles consisting of 5 min of heating to 100°Cand pressurization to 1500 psi (103 bar), 5 min static, purging,and then flushing with additional solvent.

The scutes were rinsed in deionized water prior to beingdried at 60°C for a minimum of 24 h. Lipids were extractedfrom the CNS scutes using the procedure described abovefor epidermis. We changed our laboratory procedures priorto the analysis of the CK scute samples because the mean C:N ratio of 322 loggerhead scute samples (unpublished data)was 3.3. This value is less than the 3.5 ratio suggested for lipidremoval or mathematical correction.[27] In addition, lipidextraction does not significantly alter the δ13C or δ15N valuesof epidermis.[28] Therefore, the CK scute samples were notlipid extracted. Because lipid extraction is unlikely to affectthe δ13C or δ15N values of scute, we are confident that thesamples in this study are comparable.

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Loggerhead tissue-to-tissue conversion factors

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Scute biopsies were glued ventral side down with thedorsal surface (oldest tissue) exposed, and 50 μm layers wereobtained using a carbide end mill (Sherline 5100 with digitalreadout; Vista, CA, USA). This interval was selected as thesmallest interval that would provide sufficient sample massfor stable isotope analysis. Only the newest scute layer wasused in this study. Each 50 μm layer represents approximately0.6 years of resource use.[23]

The turnover time of adult loggerhead epidermis has notbeen measured. The mean residence time of carbon andnitrogen stable isotopes in the epidermis of growing juvenileturtles is 45 and 46 days, respectively.[20] Residence times areexpected to increase with increasing body mass anddecreasing growth rates.[2] Therefore, epidermis tissueprobably represents a time period of the order of severalmonths, comparable with the time period represented in thescute subsamples used.

Stable isotope and data analysis

All samples were analyzed at the Stable Isotope Laboratoryin the Department of Geological Sciences at the Universityof Florida (Gainesville, FL, USA). Sterilized tin capsuleswere loaded with 0.5–1.0 mg of tissue and analyzed bycontinuous-flow isotope-ratio mass spectrometry usingone of two systems: (1) a DeltaPlus XL isotope ratio massspectrometer (ThermoFinnigan, Bremen, Germany) with aConFlo III interface (ThermoFinnigan) linked to a ECS 4010(Costech, Valencia, CA, USA) elemental analyzer, or (2) aDeltaV Advantage isotope ratio mass spectrometer (ThermoElectron, Bremen, Germany) coupled with a Conflo II interface(ThermoFinnigan) linked to a Carlo Erba NA 1500 CNS(Thermo Scientific, Milan, Italy) elemental analyzer.Sample stable isotope ratios relative to the isotope standard

were expressed in the following conventional delta (δ)notation: δ= ([Rsample/Rstandard] – 1) where Rsample andRstandard are the corresponding ratios of heavy to lightisotopes (13C/12C and 15N/14N) in the sample and standard,respectively. Vienna Pee Dee Belemnite was used as thestandard for 13C and atmospheric N2 for 15N. The referencematerials USGS40 (L-glutamic acid with isotopiccomposition of δ13C = –26.29 ‰ and δ15N= –4.52 ‰) andUSGS41 (L-glutamic acid enriched in 13C and 15N withisotopic composition of δ13C=37.63 ‰ and δ15N=47.57 ‰)were used to calibrate all results. The standard deviation ofUSGS40 was 0.14 ‰ for δ13C values and 0.09 ‰ for δ15Nvalues (n = 101). Along with the newest layers reported here,the entire scute records consisting of multiple layers fromeach individual were run for separate studies,[23] hence thelarge number of USGS40 standards reported. The standarddeviation of USGS41 was 0.17 ‰ for δ13C values and 0.70 ‰for δ15N values (n=13). Repeated measurements of alaboratory reference material, loggerhead scute (isotopiccomposition of δ13C=7.75‰ and δ15N= –18.45‰), were usedto ensure consistency among the results from the differentinstruments and to examine variance in a homogeneous samplewith similar isotopic composition to the samples in this study.The standard deviation for this laboratory reference materialwas 0.39‰ for δ13C values and 0.19‰ for δ15N values (n=17).The relationship between scute and epidermis was

calculated using linear regressions for both δ13C and δ15Nvalues. The C:N ratio was determined for a subset of the CK

Copyright © 2014Rapid Commun. Mass Spectrom. 2014, 28, 2059–2064

individuals for which elemental composition data wereavailable (scute: n = 17, epidermis: n = 16) by dividing %Cby %N. The C:N ratios in scute and epidermis were comparedby a Wilcoxon-Mann-Whitney test. Single-value scute-to-epidermis discrimination factors for this study werecalculated for comparison with previously published databy subtracting epidermis δ13C or δ15N values from thoseof scute. Previously published diet-tissue discriminationfactors were available for loggerhead hatchlings andjuveniles.[20] In that study, isotopic discrimination (ΔX)was estimated as δXtissue – δXdiet. The original data did notpermit the calculation of a scute-to-epidermis conversion in theform of a linear regression. Therefore, to facilitate comparisonof our data with previous loggerhead tissue studies, wecalculated a single-value scute-to-epidermis conversion factorfor each dataset (ΔXscute-epidermis =ΔXscute-diet–ΔXepidermis-diet).Statistical analyses were performed using the program R[29]

with α=0.05.

RESULTS

The linear relationship between scute and epidermis of adultloggerhead sea turtles was significant for both δ13C and δ15Nvalues ( p <0.001 for both regressions, Fig. 1). The slopebetween δ13C tissue values was not significantly differentfrom 1 ( p= 0.07), whereas the slope between δ15N tissuevalues was significantly different from 1 ( p= 0.003).Therefore, a single-discrimination factor would not beappropriate for relating δ15N values, as the offset betweentissues would change depending on the isotope values. Theintercept in the δ13C relationship was not significantlydifferent from 0 ( p= 0.76), while the intercept in the δ15Nrelationship was significantly different from 0 ( p <0.001),with δ15N values higher in epidermis than in thecorresponding scute sample.

The C:N ratio (mean± SD) of scute (3.3 ± 0.1, n = 17) wassignificantly higher than that of epidermis (3.2 ± 0.1, n = 16),although the difference was small and unlikely to bebiologically meaningful (W=255, p= 0.001).

DISCUSSION

The carbon and nitrogen isotope ratios of loggerhead scuteand epidermis were significantly related. The δ13C valuesdiffered little between the two tissues, while the δ15N valueswere higher in epidermis than in scute. The stable isotopecomposition of different tissues of the same organism can differdue to factors such as differential fractionation duringassimilation and metabolic processing, macronutrient routing,and the biochemical composition.[2,19,30] Fractionation andmacronutrient routing were described previously. Thebiochemical composition can contribute to isotopic differencesbecause each amino acid comprising the tissue proteins canvary in δ13C and δ15N values.[31,32] We cannot determine whichprocesses drive the scute-epidermis stable isotope differences,but we suspect the differences are primarily due to differingbiochemical composition. The C:N ratio can be an indicator ofthe protein content, as the nitrogen abundance is high inproteins. The C:N ratio varied little between the tissues,

wileyonlinelibrary.com/journal/rcmJohn Wiley & Sons, Ltd.

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−20 −18 −16 −14 −12 −10 −8

−20

−18

−16

−14

−12

−10

−8

Scute δ13C(‰)

y = 0.90x − 0.25

a

2 4 6 8 10 12 14

2

4

6

8

10

12

14

Scute δ15N(‰)

y = 0.87x + 2.41

b

Epi

derm

is δ

13C

(‰)

Epi

derm

is δ

15N

(‰)

Figure 1. Linear relationships between scute and epidermis:(a) δ13C values and (b) δ15N values of loggerheadssampled at two nesting beaches in Florida. The regressionequations are provided in the figure and both explain alarge portion of the variation in the datasets (δ13C r2=0.91,p <0.001 and δ15N r2=0.94, p <0.001). Dotted line depictsa 1:1 relationship.

H. B. Vander Zanden et al.

2062

indicating that the total protein amount did not differ, butmeasuring the amino acid composition of these two tissueswas beyond the scope of this study.It is important to recognize that single-value conversion

factors may lead to erroneous results if the slope of therelationship between the two tissues is not equal to 1.

Table 2. Studies that provide tissue-to-tissue conversions in th(EPI = epidermis, RBC= red blood cells)

Loggerhead tissues Life stage

Red blood cells, epidermis Adult

Scute, epidermis Adult

Mother scute, egg yolk Mother-offsprin

Mother epidermis, hatchling epidermis Mother-offsprin

wileyonlinelibrary.com/journal/rcm Copyright © 2014 John Wile

Therefore, we have provided linear relationships betweenthese two tissues and summarized other regression equationsavailable in the literature (Table 2). We encourage researchersreporting tissue-to-tissue comparisons in future studies topublish the regression results rather than single-valueconversion factors. This approach will provide the mostaccurate comparison between tissues, and it is applicable toany organism, not just sea turtles.

In order to compare our data with those from juvenile andhatchling loggerheads,[20] we also calculated single-valuediscrimination factors. Scute-to-epidermis discriminationfactors varied among the three life stages (Table 3), which islikely an effect of differences in growth and diet. Nitrogendiet-tissue discrimination factors, and, therefore, tissue-to-tissue conversion factors, are predicted to vary as a resultof the protein balance, which can be influenced by the animal’sgrowth rate and overall protein intake.[12] Less is known aboutthe effects of carbon diet-tissue discrimination factors, but diettype, including C3 vs C4 plant source, can affect carbon stableisotope discrimination factors.[19,33] The diet sources were verydifferent for these studies, as the adults were feeding in thewild, whereas the hatchlings and juveniles were maintainedin captivity on controlled diets.[20] Surprisingly, there was morevariation in the carbon scute-to-epidermis conversion factorsthan in those of nitrogen, and more research is needed onhow carbon isotope discrimination factors might be affectedby diet.

As stable isotope analysis continues to inform theconservation of sea turtles, it is important to maximize theutility of disparate datasets. The isotopic data from adultloggerhead epidermis and scute have not previously beencomparable, but the equations provided here allow isotopicdatasets using the two tissues to be combined in future analyses.Loggerhead-specific δ13C and δ15N isoscapes spanning the Gulfof Mexico were recently developed using scute stable isotopevalues, and their use was validated to accurately determine theforaging area origin of nesting females.[34] The utility of theseisoscapes to future research was limited to assigning females toorigin, based only on scute stable isotope values, but theconversion equations provided here will increase the versatilityof these isoscapes by also permitting the use of epidermissamples to determine the geographic origin of nesting femalesin this region. Tissue-to-tissue conversions allow differenttissue samples from the same species to be compared morereadily, and we underscore the importance of these conversionfactors in the form of linear regressions.

e form of linear equations between loggerhead tissue types

Equations Source

δ13CEPI = 1.00*δ13CRBC + 1.40[39]

δ15NEPI = 1.12*δ15NRBC+ 0.52δ13CEPI = 0.90*δ13CSCUTE – 0.25 This studyδ15NEPI = 0.87*δ15NSCUTE + 2.41

g δ13C equation not determined [35]δ15NYOLK = 0.73*δ15NSCUTE + 4.69

g δ13CEPI = 0.51*δ13CHATCH – 7.38 [38]δ15NEPI = 1.02*δ15NHATCH – 1.02

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Table 3. Mean loggerhead scute-to-epidermis discriminationfactors in different life stages±SD. Juvenile and hatchlingdiscrimination factors were calculated from previouslypublished data.[20] Adult data are from this study

Loggerhead lifestage

Δ13Cscute-epidermis(‰)

Δ15Nscute-epidermis(‰)

Adult �1.20 ± 0.99 �1.35 ± 0.72Juvenile 0.66 ± 0.61 �2.24 ± 0.11Hatchling �3.48 ± 0.66 �1.04 ± 0.20

Loggerhead tissue-to-tissue conversion factors

AcknowledgementsThe authors thank J. Curtis for help with stable isotopeanalysis, P. Eliazar and T. Kaufman for help with samplepreparation, and S. Good for constructive comments on themanuscript. This study was funded by National MarineFisheries Service, US Fish and Wildlife Service, DisneyWorldwide Conservation Fund, National Fish and WildlifeFoundation, Cape Canaveral National Seashore, and the SeaTurtle Grants Program. The Sea Turtle Grants Program isfunded from proceeds from the sale of the Florida Sea TurtleLicense Plate (learn more at www.helpingseaturtles.org). Allsample collections were made in compliance with theUniversity of Florida Institutional Animal Care and UseCommittee, and the Florida Fish and Wildlife ConservationCommission permits MTP-016 and 155.

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