THE FORAGING ECOLOGY OF LOGGERHEAD TURTLES IN THE NORTH ATLANTIC:

EVIDENCE FROM STABLE ISOTOPE VALUES

By

MARIELA E. PAJUELO

A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL

OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT

OF THE REQUIREMENTS FOR THE DEGREE OF

DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA

2015

© 2015 Mariela E. Pajuelo

To my ever-supporting parents, Furtiño and Rica, my wonderful husband Lucas, and sweetest

son Gabriel

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ACKNOWLEDGMENTS

I thank my advisors Drs. Karen A. Bjorndal and Alan B. Bolten, for their constant and

generous support, guidance, utmost patience, and enthusiasm throughout all these years. I feel

privileged to have been one of their students. I also thank my committee member Dr. Jeffrey A.

Seminoff who supported me from the very beginning of my graduate studies, and who along

with Drs. Bruce MacFadden and Mark Brenner helped with ideas to develop my project.

I also thank the members of the Archie Carr Center for Sea Turtle Research: Hannah

Vander Zanden, Melania López-Castro, Patricia Zárate Bustamante, Luciano Soares, Joseph

Pfaller, Robert Brown, and Peter Eliazar for their unconditional support and friendship during

the PhD program. I am very thankful to my collaborators Michael D. Arendt and his crew from

South Carolina Division of Natural Resources, Kimberly Reich, Allen Foley, Barbara A.

Schroeder, Blair E. Witherington, Pearse Webster, Robbert Prescot, Peter Dutton, Lucy A.

Hawkes, Jeffrey Seminoff, and Cynthia Lagueux for their invaluable support. I am also thankful

to Jason Curtis and the Stable Isotope Geochemistry Lab at UF, who helped with guidance

through sample processing. Undergraduates Gaithe St. Cyr, Kelseanne Breder, Laura Palomino,

Devan Patel, and Fabio Biondolillo were of tremendous help during sample preparation and

processing and for that I am very thankful to them. I am also thankful to Chelsie Papiez for

sample collection and for her friendship.

My parents Furtiño and Rica deserve a lot of gratitude for their continued support and

unconditional love all these years. My love to and from my siblings Elí, Ethel and Edgar has

always been felt present and for that I am very grateful. I also thank my best friend Jorge Lingán

for always being present for me. To my husband Lucas, I give my love and gratitude for being

the best teammate and for sharing with me and our sweet son Gabriel his passion for nature. I am

also thankful to my parents in law, Diana and Terry, for their love and support.

5

Financial support for this project was provided by Fisheries and Wildlife Foundation,

U.S. Fish and Wildlife Service, U.S. National Marine Fisheries Service, Western Pacific

Regional Fishery Management Council, the Knight Vision Foundation, Lerner Gray Fund for

Marine Research, Cynthia A. Melnick scholarship, PADI grant, UF Department travel grants, UF

College of Liberal Arts and Sciences travel grants, and UF Graduate Student Council travel

grants.

6

TABLE OF CONTENTS

page

ACKNOWLEDGMENTS ...............................................................................................................4

LIST OF TABLES ...........................................................................................................................8

LIST OF FIGURES .........................................................................................................................9

LIST OF ABBREVIATIONS ........................................................................................................11

ABSTRACT ...................................................................................................................................12

CHAPTER

1 INTRODUCTION ..................................................................................................................14

Background and Research Problem ........................................................................................14 Stable Isotopes as Tracers of Resource Use in Marine Environments ...................................15

Research Objectives ................................................................................................................17

2 DISTRIBUTION OF FORAGING HABITATS OF MALE LOGGERHEAD

TURTLES (Caretta caretta) AS REVEALED BY STABLE ISOTOPES AND

SATELLITE TELEMETRY ..................................................................................................19

Background .............................................................................................................................19 Material and Methods .............................................................................................................21

Data and Sample Collection ............................................................................................21

Sample Preparation and Analysis ....................................................................................23 Statistical analysis ...........................................................................................................24

Results.....................................................................................................................................25 Discussion ...............................................................................................................................27

Isotopic Signatures Among Different Tissues of Adult Male Loggerheads ...................27

Variation in 13

C and 15

N Among Satellite-Tracked Male Loggerheads.....................28

Relation Between Body Size and 15

N Signatures .........................................................32 Implications of the Geographic Variation of Isotopic Signatures ...................................33

Conclusions.............................................................................................................................35

3 ASSIGNMENT OF NESTING LOGGERHEADS TURTLES TO THEIR FORAGING

AREAS IN THE NORTHWEST ATLANTIC USING STABLE ISOTOPES......................45

Background .............................................................................................................................45 Material and Methods .............................................................................................................48

Data and Sample Collection ............................................................................................48 Sample Preparation and Analysis ....................................................................................51 Statistical Analyses ..........................................................................................................51

Results.....................................................................................................................................52

7

Discussion ...............................................................................................................................54

Isotopic Characterization of the Geographic Areas Used by Adult Loggerheads in

the NWA ......................................................................................................................54 Foraging Locations of Adult Female Loggerheads in the NWA ....................................58

Conservation Implications ...............................................................................................62 Conclusions.............................................................................................................................63

4 LONG-TERM RESOURCE USE AND FORAGING SPECIALIZATION IN ADULT

MALE AND FEMALE LOGGERHEAD TURTLES ...........................................................73

Background .............................................................................................................................73

Methods ..................................................................................................................................75 Data Collection ................................................................................................................75

Scute Preparation and Analysis .......................................................................................76 Data Analysis ...................................................................................................................77

Results.....................................................................................................................................78 Discussion ...............................................................................................................................80

Temporal Consistency in Resource Use ..........................................................................80 Individual Specialization .................................................................................................83

Conclusions.............................................................................................................................87

5 SUMMARY AND FUTURE RESEARCH ............................................................................94

Summary .................................................................................................................................94

Significance and Implications .................................................................................................96

Future Research ......................................................................................................................97

APPENDIX EPIDERMIS AND PLASMA ISOTOPIC VALUES ...........................................100

LIST OF REFERENCES .............................................................................................................101

BIOGRAPHICAL SKETCH .......................................................................................................114

8

LIST OF TABLES

Table

page

2-1 Foraging area, sample size (N), and body size (SCL) of the five groups determined

according to the migration that satellite-tracked male loggerheads followed after the

mating season. ....................................................................................................................36

2-2 Adult male loggerhead population range (maximum – minimum) and variance (Var)

of 13

C and 15

N values in red blood cells (RBC), epidermis (EPI), and plasma

(PLA) samples. ..................................................................................................................37

3-1 Location (state, breeding/foraging area, and latitude), year of collection, and sample

size of epidermis samples from adult loggerhead turtles with known and unknown

foraging grounds used in this study. ..................................................................................65

3-2 Assignment of adult female loggerheads of unknown foraging location to a

geographic area with ≥ 80% probability of group membership. Values in parentheses

are additional turtles assigned with a probability < 80% of group membership. ..............66

4-1 Mean and range of carapace length for individual loggerhead turtles, and mean and

total range of 15N and 13C values of loggerhead turtle scute tissue sampled at two

foraging areas, South Carolina/Georgia (SC/GA) and Florida Bay (FLB). For isotope

values, mean is the mean range of isotopic values within individual turtles and total

is the range of isotopic values across all individuals. ........................................................89

9

LIST OF FIGURES

Figure page

2-1 Stable isotope ratios (13

C and 15

N) of adult female and male loggerheads in

Florida. ...............................................................................................................................38

2-2 Spatial distribution of satellite-tracked male loggerheads from Cape Canaveral,

Florida after mating season in April 2006 and 2007. .........................................................40

2-3 Linear relationships between epidermis and red blood cells for 13

C and 15

N of

adult male loggerheads.. ....................................................................................................41

2-4 Stable isotope ratios of carbon and nitrogen of red blood cells, epidermis, and plasma

from adult male loggerheads collected at Cape Canaveral, FL, versus the latitude to

which turtles migrated after the mating season..................................................................42

2-5 Red blood cell 15

N versus straight carapace length in male loggerhead turtles that

remained off Cape Canaveral.............................................................................................43

2-6 Comparison of stable isotope ratios of 13

C and 15

N of food web organisms at the

different foraging locations visited by male loggerheads after the mating season. ...........44

3-1 Distribution of stable isotope ratios (13

C and 15

N) of adult male and female

loggerheads in the Northwest Atlantic. ..............................................................................67

3-2 Map showing the locations of the six nesting areas and single foraging ground

sampled in this study: Bald Head Island, Wassaw Island, Sapelo Island, Blackbeard

Island, Jekyll Island, Cumberland Island, and Florida Bay.. .............................................68

3-3 Stable isotope ratios (13

C and 15

N) of adult female loggerheads in the Northwest

Atlantic. ..............................................................................................................................69

3-4 Relationship between carbon and nitrogen stable isotope values of adult female

loggerheads and the latitude to which they migrated after the nesting season. .................70

3-5 Stable isotope ratios of carbon and nitrogen of adult loggerhead turtles with known

foraging location showing three groups representing the three geographic areas used

by adult loggerheads in the Northwest Atlantic: Mid-Atlantic Bight, South Atlantic

Bight, and Subtropical Northwest Atlantic. .......................................................................71

3-6 Breeding population structure according to foraging area used by loggerheads

nesting along the U.S. Atlantic coast as determined through discriminant analysis

using carbon and nitrogen stable isotope values of adult loggerhead turtles with

known foraging grounds as reference data.. ......................................................................72

4-1 Foraging locations, South Carolina/Georgia and Florida Bay, where male

loggerheads turtles were sampled for scute tissue.. ...........................................................90

10

4-2 Resource use of individual male loggerhead turtles as indicated by 15

N and 13

C

values of successive scute layers. ......................................................................................91

4-3 Thickness of scute in relation to straight carapace length in loggerhead turtles from

South Carolina/Georgia and Florida Bay. ..........................................................................92

4-4 Comparison of temporal consistency and degree of individual specialization between

loggerhead turtles from two foraging areas, South Carolina/Georgia and Florida

Bay. ....................................................................................................................................93

A-1 Stable isotope ratios (13

C and 15

N) of epidermis and plasma samples of adult male

loggerheads in Florida......................................................................................................100

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LIST OF ABBREVIATIONS

BIC Between individual component

CSIA-AA Compound-specific stable isotope analysis of amino acids

EPI Epidermis

GoM Gulf of Mexico

MAB Mid-Atlantic Bight

MANOVA Multivariate analysis of variance

NWA Northwest Atlantic

PLA Plasma

POM Particulate organic matter

RBC Red blood cells

SAB South Atlantic Bight

SCL Straight carapace length

SI Stable Isotopes

SNWA Subtropical Northwest Atlantic

TNW Trophic niche width

WIC Within individual component

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Abstract of Dissertation Presented to the Graduate School

of the University of Florida in Partial Fulfillment of the

Requirements for the Degree of Doctor of Philosophy

THE FORAGING ECOLOGY OF LOGGERHEAD TURTLES IN THE NORTH

ATLANTIC: EVIDENCE FROM STABLE ISOTOPE VALUES

By

Mariela E. Pajuelo

December 2015

Chair: Karen A. Bjorndal

Major: Zoology

Sea turtles spend the majority of their lives in the marine environment but are more easily

accessible at their nesting beaches. Thus, understanding the relationships between their various

foraging grounds and breeding areas is essential to assess population dynamics. In the particular

case of the Northwest Atlantic (NWA) population, its life history stages and the habitats it

occupies have been identified. What still remains poorly understood is the role loggerheads play

within their ecosystems. Understanding the foraging ecology of loggerheads is necessary for

revealing this role. The goal of this study was to further our understanding of the foraging

ecology of loggerhead turtles in the North Atlantic Ocean using stable isotope analysis.

First, I investigated the foraging habitats of highly elusive male loggerhead turtles using

stable isotopes and satellite telemetry data. Male loggerhead isotopic data varied with foraging

location and this is explained by geographic isotopic variation at the base of the food web. Also,

comparison of male loggerhead isotopic values with those of female loggerheads revealed that

males may exhibit similar foraging strategies (diet and habitat use) to those of females. Next, I

characterized isotopically the foraging regions for loggerhead turtles in the NWA using a

combination of satellite telemetry and stable isotope analysis, thus validating the use of stable

isotope analysis to identify foraging areas of loggerhead turtles in the NWA. The largest

13

assignment of nesting loggerheads to their foraging locations revealed that turtles segregate

geographically in their use of foraging areas. I also examined the long-term consistency in

resource use and degree of foraging specialization in male loggerheads. Individual male

loggerheads exhibit a specialized foraging behavior similar to females that is consistent for up to

17 years. My results also revealed that resource diversity has an effect on the degree of

individual specialization in loggerhead turtles.

In summary, this study expanded our knowledge of loggerhead foraging strategies and

demonstrated that carbon and nitrogen stable isotopes of loggerhead turtles are effective

biochemical tags to link loggerhead foraging grounds and breeding areas.

14

CHAPTER 1

INTRODUCTION

Background and Research Problem

As widely distributed marine organisms, species of sea turtles are composed of

populations that can vary in size, geographic distribution, and population dynamics, and some

populations of sea turtles have shown dramatic long-term changes in population numbers. A

clear example is the loggerhead sea turtle (Caretta caretta) nesting population in the Northwest

Atlantic (NWA), one of the largest nesting aggregations for the species in the world (Ehrhart et

al. 2003), whose numbers declined markedly from 1998 to 2007 (Witherington et al. 2009) but

have increased steadily ever since (Arendt et al. 2013).

Sea turtles have complex life histories, with ontogenetic diet and habitat shifts, long

migrations to developmental habitats and breeding areas, and fidelity to their nesting areas and

foraging grounds. While most studies of sea turtles have been conducted on nesting beaches

where female turtles are more easily accessible, sea turtles spend the majority of their lives in the

marine environment. Thus, understanding the relationships among the various developmental

and foraging areas to their breeding areas is essential to accurately assess population dynamics,

which in turn will help improve the design of management strategies for the conservation of sea

turtle populations.

In the case of the NWA population, its life history stages and the habitats it occupies have

been identified (Bolten 2003). After hatching in beaches along the U.S. east coast, hatchlings

embark on one of their first long migrations to oceanic developmental habitats across the

Atlantic Ocean (Bolten 2003), where they forage and grow for 7–15 years (Avens et al. 2013).

Juvenile loggerheads migrate from oceanic habitats to neritic areas where the majority will

remain, but some juvenile loggerheads still rely on the use of oceanic waters (McClellan et al.

15

2007; Mansfield et al. 2009). In any case, NWA adult loggerhead turtles exclusively use coastal

foraging areas (Hawkes et al. 2011; Ceriani et al. 2012; Foley et al. 2014; Griffin et al. 2014) that

are widely distributed along the U.S. east coast, the Gulf of Mexico, the Bahamas, and Cuba.

What still remains poorly understood is the role loggerhead turtles play within their

ecosystems. Furthering our understanding of the foraging ecology of loggerheads will allow us to

better understand this role, as a primary way that organisms interact with other species and with

their environment is through their diet (Bjorndal 2003). Loggerhead turtles use a wide range of

foraging areas with distinct biotic and abiotic factors that could potentially have an effect on

their foraging strategies, which in turn can have profound effects on the demographic parameters

of populations.

This research focuses on understanding the foraging strategies, including diet, habitat use

and foraging area use of loggerhead turtles in the North Atlantic Ocean using stable isotope

analysis.

Stable Isotopes as Tracers of Resource Use in Marine Environments

Stable isotope analysis is a technique that uses ratios of stable isotopes of naturally

occurring elements. In the marine environment, carbon and nitrogen stable isotopes (13

C and

15

N, respectively) have been widely used to evaluate the foraging and migratory ecology of

highly migratory marine animals (Schell et al. 1989; Jaeger et al. 2010; McKenzie et al. 2011;

Zbinden et al. 2011; Pajuelo et al. 2012a,b; Lorrain et al. 2012; Seminoff et al. 2012). This is

based on the fact that the stable isotope composition of an organism’s tissue reflects that of the

food or nutrients assimilated at its foraging area and that such stable isotope composition can

vary geographically because of biogeochemical processes (Hobson 1999).

16

During assimilation, there is a stepwise increase of 15

N between the diet and consumer

such that 15

N values are typically used to estimate an organism’s trophic position (DeNiro and

Epstein 1981; Post 2002). However, discrimination factors in 13

C (the difference in 13

C

between diet and consumer) are smaller (DeNiro and Epstein 1978) and vary with primary

production (Michener and Kaufmann 2007) and habitat type: pelagic versus benthic or oceanic

versus neritic (Hobson et al. 1994). Thus, 13

C has been mainly used to identify carbon sources

and habitats utilized (Hobson et al. 1994). Ultimately, the 13

C and 15

N values of a marine

consumer are not just a function of the isotopic values of its diet, but also the isotopic values of

primary producers at the base the food web (Schell et al. 1989; Minami and Ogi 1997; Burton

and Koch 1999; Cherel and Hobson 2007; Pajuelo et al. 2010), which can vary with geographic

location as a result of biogeochemical processes (Goericke and Fry 1994; Montoya 2007;

McMahon et al. 2013).

Nitrogen fixation and denitrification are two of the main processes by which nitrogen

cycles in the marine environment and that affect the 15

N composition of primary producers at

the base of the food web (e.g., marine phytoplankton, seagrass, algae). Nitrogen fixation lowers

and denitrification increases the 15

N values of primary producers (Montoya 2007). Additionally,

13

C values of marine phytoplankton and particulate organic matter (POM, a proxy for primary

production) decrease from the equatorial zones toward the polar regions (Goericke and Fry 1994)

as a result of differential plankton growth, sea water temperature, and dissolved CO2 (Goericke

and Fry 1994; Graham et al. 2010). These isotopic differences at the base of the food web create

isotopically distinct areas (McMahon et al. 2013) that allow us to use 13

C and 15

N to

distinguish foraging areas used and assess residency and migration patterns of marine organisms

(Graham et al. 2010). Therefore, it is essential to understand how baseline isotopic values vary

17

across the range of foraging areas used by highly migratory organisms before using stable

isotopes to make comparison between populations using distant foraging areas.

Research Objectives

This study aimed to improve our understanding of the foraging ecology of loggerhead

turtles in the North Atlantic Ocean. My primary research objectives were to: 1) use stable isotope

composition to investigate the foraging habitats of adult male loggerheads, 2) identify key

foraging areas of loggerheads using stable isotope analysis and assign nesting loggerheads to

these areas, and 3) assess temporal trends in resource use and degree of individualization in

resource use in adult male and female loggerheads.

These objectives are addressed and discussed in the following 4 chapters. In Chapter 2, I

provide insights into the foraging habitats of elusive male loggerhead turtles sampled at one

breeding area in the NWA using stable isotope analysis. I compared the stable isotope values of

male loggerheads with those previously reported for female loggerheads in the region, finding

that males may also exhibit foraging strategies (diet and habitat use) similar to those of female

loggerheads. Male turtles were satellite tracked after sampling for stable isotopes and followed

back to their foraging locations. Thus, I reveal how loggerhead isotopic values vary with

foraging location, which can be explained by differences in isotopic values at the base of the

food web rather than differences in loggerhead’s trophic levels. Chapter 2 has been published in

the journal Marine Biology (Pajuelo M, Bjorndal KA, Reich KJ, Arendt MD, Bolten AB (2012)

Distribution of foraging habitats of male loggerhead turtles (Caretta caretta) as revealed by

stable isotopes and satellite telemetry. Marine Biology 159:1255–1267).

In Chapter 3, I characterize isotopically the main foraging regions for loggerheads in the

NWA using a combination of satellite telemetry and stable isotope analysis, thus validating the

use of stable isotope analysis to identify foraging areas of loggerhead turtles in the NWA. This

18

allowed me to assign a large number of female loggerhead turtles from various nesting beaches

to their foraging locations using stable isotope values alone, finding that loggerhead turtles

segregate geographically in their use of foraging areas. Chapter 3 has been published in

Ecosphere (Pajuelo MA, Bjorndal KA, Reich KJ, Vander Zanden HB, Hawkes LA, Bolten AB

(2012) Assignment of nesting loggerhead turtles to their foraging areas in the Northwest Atlantic

using stable isotopes. Ecosphere 3:art89, doi:10.1890/ES12-00220.1).

In Chapter 4, I evaluate the long-term consistency in resource use and degree of

individual foraging specialization in male loggerhead turtles. Using stable isotopes as a proxy for

diet and habitat use, I reaffirm that long assumed generalist loggerhead turtles are composed of

individuals with specialized foraging behaviors. The wide isotopic variation at the population

level revealed that male loggerheads are part of a generalist population, while the small variation

in isotopic values within individual male turtles compared to that of the population revealed that

males exhibit specialization in diet and habitat use. Similar to studies in female loggerhead and

green turtles (Chelonia mydas), this specialized foraging behavior in male loggerheads is

maintained consistently over time. I also present initial results on the effect of resource diversity

on the individual specialization in male and female loggerhead turtles. Chapter 4 will be

submitted to Marine Biology under the authorship of Pajuelo M, Bjorndal KA, Arendt MD,

Foley AM, Schroeder BA, Witherington BE, and Bolten AB. Finally, in Chapter 5, I provide a

summary of my research to date, discuss its significance, and suggest areas for further research.

19

CHAPTER 2

DISTRIBUTION OF FORAGING HABITATS OF MALE LOGGERHEAD TURTLES

(Caretta caretta) AS REVEALED BY STABLE ISOTOPES AND SATELLITE TELEMETRY1

Background

Knowledge of foraging ground distribution of highly migratory animals is critical for

understanding their foraging behavior and habitat use. Identification of key habitats not only

helps to characterize life history features of populations (Block et al. 2001), but also to assess the

impact of threats that populations may face (Hays et al. 2003). Most efforts to identify key

habitats and movement patterns have used flipper tags (Limpus et al. 1992), genetic markers

(Bolker et al. 2007), chemical analysis (Thorrold et al. 2001), and electronic tagging (Block et al.

2005; Hawkes et al. 2011). Electronic tagging is an excellent tool for assessing the movement

and foraging behavior of marine animals, but this tool is constrained to small sample sizes due to

expense and to organisms large enough to be tagged (but see Block et al. 2005). Additional

information on migration patterns and foraging habitat use of highly migratory and elusive

marine animals can be obtained using stable isotopes (SI) (Reich et al. 2007; Rooker et al. 2008;

Newsome et al. 2009).

In marine ecosystems, δ15

N values vary predictably with trophic level (Minagawa and

Wada 1984), while δ13

C values vary with source of primary production (Michener and

Kaufmann 2007) and habitat type: pelagic versus benthic or oceanic versus neritic (Hobson et al.

1994). Additionally, both δ15

N and δ13

C may vary with geographical location as a result of the

effect of oceanic processes on baseline δ15

N and δ13

C (Goericke and Fry 1994; Montoya et al.

Reprinted with permission from the Publisher. The final publication is available at link.springer.com. Original

publication: Pajuelo M, Bjorndal KA, Reich KJ, Arendt MA, Bolten AB (2012a) Distribution of foraging habitats of

male loggerhead turtles (Caretta caretta) as revealed by stable isotopes and satellite telemetry. Mar Biol

159:1255−1267

20

2007), which in turn are reflected in higher trophic level organisms (Cherel and Hobson 2007;

Pajuelo et al. 2010). Marine phytoplankton and particulate organic matter δ13

C values decrease

from the equatorial zones toward the polar regions (Goericke and Fry 1994) as a result of

differential plankton growth rates, dissolved CO2 concentration in seawater, and seawater

temperature, among other factors (Goericke and Fry 1994; Graham et al. 2010). Characteristic

nitrogen cycle regimes—nitrogen fixation and denitrification—lower and increase, respectively,

the δ15

N of primary producers (Montoya 2007). These baseline isotopic differences create

isotopically distinct regions, which can then be used to assess movements of individuals

migrating among them (Graham et al. 2010). Therefore, it is important to know the community

baseline δ15

N and δ13

C when identifying foraging habitats in highly migratory organisms.

Loggerhead sea turtles (Caretta caretta) are listed as Endangered on the IUCN Red List

(IUCN 2011). Understanding the foraging ecology and movement patterns of loggerheads

improves their conservation outlook. While several studies have addressed various aspects of the

foraging ecology of adult female and juvenile loggerheads in the North Atlantic (Hawkes et al.

2007, 2011; Wallace et al. 2009; Frick et al. 2010; McClellan et al. 2010; Vander Zanden et al.

2010), little is known about the foraging strategies of adult male loggerhead turtles (but see

Arendt et al. 2012a, 2012b).

Recent work by Reich et al. (2010) revealed a large range in the δ15

N and δ13

C signatures

of skin samples in nesting loggerheads in Florida, USA. Two clusters were identified based on

δ13

C signatures that were consistent with differences in habitat use (oceanic versus neritic waters;

Figure 2-1a). However, the authors could not rule out the possibility of other factors affecting

carbon signatures, such as geographical location or differential source of primary production.

Furthermore, even though the range from 2 to 15‰ in δ15

N of females was not assessed, this

21

could represent differences in trophic levels (Post 2002) or baseline isotopic values (Pajuelo et

al. 2010). Recent observations of migration patterns of adult male loggerheads in Florida through

satellite telemetry have revealed the use of different geographic foraging areas after the mating

season (Figure 2-2; Arendt et al. 2012a, 2012b). Do adult male loggerheads in Florida exhibit a

pattern in SI values similar to that in nesting loggerheads? If so, how do patterns in δ15

N and

δ13

C of male loggerheads compare with their satellite tracking data? To answer these questions I

collected tissues from 29 satellite-tracked adult male loggerheads at one breeding area in Florida

and analyzed them for δ15

N and δ13

C. Tissues from additional male turtles not fitted with satellite

transmitters were used to compare SI patterns between adult males and females and to assess SI

values of different tissues within individual males. By integrating SI with satellite telemetry, I

seek to reveal the foraging strategy of adult male loggerheads in the Northwest Atlantic.

Material and Methods

Data and Sample Collection

Thirty-seven adult male loggerhead turtles (straight carapace length, SCL >86 cm) were

captured by trawling from the Port Canaveral shipping channel, Florida, USA (28.38N,

80.53W) during mating season in April 2006 and 2007. Sexual maturity of each turtle was

confirmed by laparoscopy (Blanvillain et al. 2008). All turtles were used to evaluate SI values

within and among adult male loggerhead tissues, while 29 turtles were used to assess turtle

movements by combining satellite telemetry and SI data.

Satellite transmitters (ST-20, Model A2020; Telonics, Inc., Mesa, Arizona, USA) were

attached to 29 males (see Arendt et al. 2012a), and their movements were tracked during and

after the mating season. Arendt et al. (2012a, 2012b) characterized the distinct movement

patterns of these males that I classify here into five groups: (1) residency in waters near the

22

breeding area in Cape Canaveral, FL, (2) northern migration and residency in waters off South

Carolina in the South Atlantic Bight, (3) northern migration to foraging grounds along the

continental shelf from North Carolina to New Jersey in the Mid-Atlantic Bight (MAB), (4)

southern migration to shallow waters of the Florida Keys and the Bahamas in the subtropical

NWA, and (5) southern migration and ultimate residency in coastal waters of the northeast Gulf

of Mexico (Figure 2-2). I grouped turtles based on their migration patterns and foraging locations

with similar oceanographic conditions.

Body size (SCL) was recorded using tree calipers marked with 0.1 cm units, and blood

samples were collected for each adult male loggerhead. Epidermis (EPI) samples were also

collected for 26 turtles, 20 of them corresponding to satellite-tracked turtles. Only one turtle was

recaptured in consecutive years, and blood samples were collected in both years.

Epidermis samples (N = 26) were collected from the dorsal surface of the neck using a 6 mm

biopsy punch. Blood samples were collected from the dorsal cervical sinus (Owens and Ruiz

1980) using a vacutainer tube with sodium heparin, fitted with a 21-gauge needle. Sodium

heparin does not affect isotopic values (Lemons et al. 2012). Blood was centrifuged for 5 min

within 1 h of collection and separated into red blood cells (RBC, N =38) and plasma (PLA, N =

38), which were stored in cryovials. All samples were stored frozen until dried at 60C prior to

sample preparation and analysis. EPI and RBC reflect the turtle’s dietary history over a longer

period of time (at least 4.2 months) than PLA (at least 2 months) based on studies conducted

with growing juvenile loggerheads (Reich et al. 2008). Isotope turnover (i.e., the time the

isotopic composition in the consumer tissue reaches equilibrium after a shift in resource use) for

adult loggerheads may be longer because rates of isotopic incorporation slow with reduced

23

growth rates (Reich et al. 2008), and because these rates are allometrically dependent on body

mass (Carleton and Martínez del Rio 2005).

To evaluate turtle movements, turtles from Group 1 (resident males) were only included

if they stayed near Cape Canaveral for at least 60 days, well after all migrants left the breeding

area. Turtles from Groups 2 through 5 (migratory males) were included if transmissions lasted a

minimum of 30 days at the foraging ground. Following these criteria, movements of 4 turtles

could not be determined because transmissions failed before the 30 or 60 day cut offs, and a total

of 25 turtles (Table 2-1) was used to relate SI with satellite telemetry data. I used the median

latitude where turtles occurred at the foraging grounds to evaluate the relationship between SI

and geographic location. Location data were extensively filtered (see Arendt et al. 2012a, 2012b

for details). Additionally, I compiled isotopic data from the literature on lower trophic-level

organisms from the geographic areas where male loggerheads traveled after the mating season.

Sample Preparation and Analysis

Turtle EPI samples were washed with deionized water and alcohol swabs to remove

epibionts and extraneous particles. The outermost layer of the turtle epidermis was separated

from the underlying tissue, finely diced with a scalpel blade, and dried at 60C for 24 h. Blood

samples (RBC and PLA) were dried for 24 h at 60C and then ground to a fine powder using a

mortar and pestle. Lipids were extracted from EPI samples with petroleum ether using an

accelerated solvent extractor. Lipids were not extracted from RBC and PLA samples because,

for these tissues, C:N ≤ 3.5. According to Post et al. (2007) no extraction of lipids is necessary

when tissue C:N < 3.5. Lipids were extracted from EPI samples to allow for comparison with

previously published EPI isotopic data.

24

For stable isotope analysis, approximately 500 to 600 μg of each sample was weighed

and sealed in a tin capsule. Samples were analyzed for δ13

C and δ15

N by combustion in a

COSTECH ECS 4010 elemental analyzer interfaced via a Finnigan-MAT ConFlow III device to

a Finnigan-MAT DeltaPlus XL isotope ratio mass spectrometer in the Stable Isotope

Geochemistry Lab at the University of Florida, Gainesville, USA. Results are presented as stable

isotope ratios of a sample relative to an international standard and reported in the conventional

notation: X = [(Rsample/Rstandard) –1] x 1000, where X is the relative abundance of 13

C or 15

N in

the sample expressed in parts per thousand (‰); Rsample and Rstandard are the ratios of heavy to light

isotope (13

C/12

C and 15

N/14

N) in the sample and international standard, respectively. The standard

used for 13

C was Vienna Pee Dee Belemnite and for 15

N was atmospheric N2. Working standards

L-glutamic acid USGS40 (13

C = -26.39‰ and 15

N = - 4.52‰) were calibrated monthly against

international standards and were inserted in all runs at regular intervals to calibrate the system.

In addition, a loggerhead scute standard (13

C = -18.36‰ and 15

N = 7.68‰) was used in all

runs. The analytical accuracy of my measurements—calculated as the SD of replicates of

standards—was 0.11 and 0.12‰ for 13

C and 15

N of working standards (N = 29), respectively,

and 0.12 and 0.16‰ for 13

C and 15

N of scute standard (N = 10), respectively.

Statistical analysis

Levene’s test was used to assess homogeneity of variances of 13

C and 15

N among the

three tissues sampled. The relationships between the isotopic signatures of the two tissues with

similar temporal isotopic assimilation, EPI and RBC, were evaluated with linear regressions. To

explore the effect of geographical location on 13

C and 15

N, the correlation between isotopic

signatures and the latitude of the foraging grounds of the turtles was evaluated using Spearman

rank test. Additionally, Wilcoxon rank sum test was used to assess body size differences between

25

two main migration patterns, northern (Group 3) versus southern (Group 4). Body size

differences among remaining Groups (1, 2, and 5) were not assessed because samples sizes were

too small (N < 3), or because turtles did not migrate to a different geographic area after the

mating season. Finally, the relationship between body size and isotopic signatures of males

within a foraging area was evaluated using linear regression whenever sample size allowed. All

data were analyzed using program R (R Development Core Team 2009) with an level of 0.05.

Results

The ranges (the difference between maximum and minimum values) of isotopic

signatures for each tissue from all adult male loggerhead samples varied between 7.53 and

8.19‰ for 13

C, while 15

N ranges varied from 8.96 to 9.68‰ (Table 2-2). The variance of

isotopic values was similar among tissues for both 13

C (Levene’s test, F = 0.19, P = 0.827) and

15

N (Levene’s test, F = 0.58, P = 0.562) (Table 2). Also, visual inspection of Figure 1b reveals a

pattern in the SI values of male turtles similar to that of female loggerheads.

When EPI and RBC—tissues reflecting the turtle’s longer-term foraging history—were

compared, I found that RBC had lower values for both 13

C and 15

N among all turtles with both

tissues sampled. Furthermore, there was a significant positive relationship between RBC and EPI

samples for both 13

C (Linear regression, r2 = 0.96, F1,24 = 623.8, P < 0.001; Fig 3a) and

15N (r

2

= 0.98, F1,24 = 1208, P < 0.001; Figure 2-3b). However, I should be cautious when using the 13

C

correction factor to obtain specific carbon isotopic values, as the data distribution is unequal

(Figure 2-3a).

I analyzed three different tissues collected from satellite-tracked loggerheads, but my

conclusions about turtle movements will be based on RBC because this was the only tissue

available for all turtles sampled that reflect the longer-term foraging history of the turtle prior to

26

capture. Male turtles that migrated south to the Bahamas and the Florida Keys had the highest

13

C and lowest 15

N values (Figure 2-1b). In contrast, the lowest values of 13

C were found in

males that migrated northward to New Jersey, while the highest 15

N value was found in a turtle

that established residency in Maryland (Figure 2-1b). A strong negative correlation was found

between the latitude of the residential foraging location and the 13

C RBC values (Spearman

rank correlation, rs = -0.73, N = 25, P < 0.001; Fig 2-4Aa). Similarly, a strong but positive

correlation was found between the latitude and the 15

N RBC values (rs = 0.78, N = 25, P <

0.001; Fig 2-4Ba). Moreover, EPI and PLA had similar correlation patterns as that of RBC. EPI

and PLA 13

C values were correlated negatively with latitude (EPI: rs = -0.77, N = 17, P < 0.001;

PLA: rs = -0.82, N = 25, P < 0.001; Fig 2-4Ab, 2-4Ac), and 15

N was positively correlated with

latitude (EPI: rs = 0.86, N = 17, P < 0.001; PLA: rs = 0.75, N = 25, P < 0.001; Fig 2-4Bb, 2-4Bc).

Only one male turtle (from Group 1) was recaptured in Port Canaveral in consecutive years and

had similar RBC 13

C (-15.57 and -15.55‰) and 15

N (10.95 and 10.80‰) values in both years.

Body size had a significant negative relationship with 15

N (Linear regression: r2 = 0.62,

F1,8 = 15.7, P = 0.004; Figure 2-5), but not with 13

C (Linear regression: r2 = 0.047, F1,8 = 1.445,

P = 0.264) in the turtles that remained near Cape Canaveral (Group 1). Sample size in other

foraging areas was too small to analyze a relationship between body size and isotopic signatures.

Additionally, body sizes were not significantly different between turtles with northernmost

foraging grounds (Group 3: using waters in the MAB) and turtles using southernmost foraging

areas (Group 4: using waters in the subtropical NWA) (Wilcoxon rank sum test, W = 7, N1 = 3,

N2 = 8, P = 0.376). However, sample sizes between north and south were unequal with only 3

turtles migrating to southernmost foraging areas (Figure 2-2).

27

Discussion

Isotopic Signatures Among Different Tissues of Adult Male Loggerheads

I found that the isotopic values of male tissues reflecting different temporal integration of

diet and habitat use (RBC, EPI, and PLA) were similar in range and variance. RBC and EPI

isotopic values were expected to be similar in range and variation because these tissues provide

the turtle’s longer-term dietary information. On the other hand, PLA reflects a relatively more

recent foraging history of a turtle (up to at least 2 months). Therefore, if males had been feeding

in waters off Cape Canaveral when captured, then PLA signatures would have been similar

among turtles. The fact that PLA samples presented a similar pattern as RBC and EPI samples

indicates that either 1) turtles had not spent enough time to allow the Cape Canaveral isotopic

signature to be incorporated, or 2) turtles had not been feeding in the breeding area.

The significant positive relationship between 13

C and 15

N of RBC and EPI (Figure 2-

3), allows me to predict EPI signatures for a given RBC sample and vice versa. EPI samples are

more easily collected than RBC samples. Correction factors of this nature can be useful when

trying to compare isotopic values among individuals and the same tissues are not available. In

this study, these correction factors allowed for general comparison between male and female

turtles. However, a systematic experiment in captivity in which turtles are consistently fed a diet

with known isotopic signatures would be the ideal way to evaluate isotopic discrimination

among different tissues, especially when specific tissue isotopic values are required to estimate

diet composition. Few such experiments have been conducted in sea turtles (Seminoff et al.

2006, 2009; Reich et al. 2008; Vander Zanden et al. unpubl. data), and no such experiments have

been conducted in adult loggerheads.

28

Variation in 13

C and 15

N Among Satellite-Tracked Male Loggerheads

In this study I found a significant relationship between both the 13

C and 15

N of male

loggerheads and the latitude of their foraging grounds. The ranges of isotopic values were 8.2‰

and 8.9‰ for RBC 13

C and 15

N, respectively, over a range of 16° latitude (Figure 2-4Aa, 2-

Ba). Large differences in the isotopic signatures of turtles in the NWA could be attributed to

differences in (1) trophic level (based on 15

N), (2) habitat type (i.e., pelagic versus benthic or

oceanic versus neritic, based on 13

C) and/or (3) geographical location (based on both 13

C and

15

N).

Turtle–diet isotopic discrimination factors for 15

N are not available for adult loggerheads.

However, given the discrimination factor of 2.5‰ for RBC in adult green turtles, Chelonia

mydas (Vander Zanden et al. 2012), the observed range in 15

N values in male loggerheads

would imply a variation of approximately 3.6 trophic levels in male loggerheads, if nitrogen

baseline signatures were equal across all foraging areas. Such trophic level differences are

unlikely to occur in NWA loggerhead turtles because they are known to prey mainly on benthic

invertebrates in coastal waters (see references cited in Hopkins-Murphy et al. 2003). A historic

shift in diet from horseshoe crabs to crustaceans, and then to mostly fish has been recently

reported in loggerheads in Chesapeake Bay, Virginia, USA, through analysis of stomach

contents; however, no turtle with SCL > 90 cm showed this diet change (Seney and Musick

2007). Male loggerhead RBC 15

N signature (mean ± SD = 12.3 ± 1.70‰) is higher than 15

N of

horseshoe crabs and blue crabs (10.3 and mean ± SD = 11.5 ± 2.40‰, respectively; Knoff et al.

2001) and lower than those of fish (range: 13.9 to 18.0‰; Buchheister and Latour 2011), which

suggests that adult male loggerheads in this region may be relying more on benthic invertebrates

than on fish.

29

The second possible explanation relates the large variation in 13

C to differential habitat

use. Because all male loggerheads dispersed to coastal locations (Figure 2-2), the variation in

13

C may be reflecting a pelagic versus benthic habitat use in neritic waters, which would result

in low versus high 13

C values, respectively. Indeed, lowest values of 13

C were found in turtles

that migrated to high latitude areas (New Jersey; Figure 2-1b), where turtles used deeper waters

(mean water depth = 28 m; M. Arendt, unpubl. data) contrasted with high 13

C turtles that

migrated to lower latitude foraging areas (Bahamas and Florida Keys; Figure 2-1b) and used

shallow waters (mean water depth = 8 m; M. Arendt, unpubl. data). Diving data for two northern

turtles revealed use of both bottom and surface waters suggesting that these feed throughout the

water column (M. Arendt, unpubl. data). However, low 13

C signatures were also present in

turtles using shallow waters in North and South Carolina (mean water depth = 5 and 7 m,

respectively; M. Arendt, unpubl. data) (Figure 2-1b). Reich et al. (2010) found similar results in

some nesting loggerheads in Florida which presented low 13

C values as well as neritic/benthic

epibionts suggesting use of shallow waters. Thus, although I cannot rule out the pelagic versus

benthic foraging strategy in male loggerheads, I propose that 13

C signatures are being affected

primarily by other factors.

The third possible explanation for the large variation of 13

C and 15

N is geographical

location. The differences in 15

N and 13

C at the base of the food web are conserved through

higher trophic levels (Cherel and Hobson 2007; Pajuelo et al. 2010). Thus, if baseline signatures

change with geographical area then the isotopic differences observed in males would reflect the

location of the foraging area rather than differences in diet or habitat use.

Indeed, different oceanographic processes and nutrient sources influence the baseline

signatures of the foraging areas used by male loggerheads. In the NWA, nitrogen fixation, which

30

lowers the 15

N signature of primary producers, is highest in the tropical and subtropical NWA

(Montoya et al. 2002). On the other hand, highly productive coastal waters near estuaries in the

Mid-Atlantic Bight are characterized by high 15

N values in primary producers apparently due to

the high 15

N contribution from human sources to these waters (McKinney et al. 2010). Also,

denitrification, a process that increases values of 15

N (Montoya et al. 2007), has been reported

in the MAB (Fennel et al. 2006). To what extent denitrification affects the 15

N of coastal biota

of the MAB has not yet been assessed (McKinney et al. 2010).

Therefore, I would expect male loggerheads foraging from Virginia to New Jersey to

have the highest 15

N signatures, while turtles foraging in areas with high rates of N2 fixation

(e.g., the Bahamas) will present the lowest 15

N signatures. This clearly corresponds with the

increase of male 15

N with latitude (Figure 2-4B), but does not necessarily support a latitudinal

effect on coastal waters from equator to polar regions because 15

N of primary producers slowly

decreases at latitudes north of the Delaware estuary (McKinney et al. 2010). The probable human

influence revealed in the 15

N signatures of males in northern waters corresponds with the

elevated concentrations of persistent organic pollutants recently found in the same male

loggerheads (Ragland et al. 2011).

Carbon isotope signatures can also reflect geographical location. High latitude primary

producers have much lower 13

C than primary producers at lower latitudes (Goericke and Fry

1994). Water temperature has recently been proposed as a proxy for baseline 13

C values

because it affects plankton growth rates and dissolved CO2 concentrations in seawater—which in

return have an effect on baseline 13

C values—(Mackenzie et al. 2011), and could explain the

13

C latitudinal gradient. This latitudinal gradient in 13

C agrees with the lowest male 13

C value

31

found in cooler waters off New Jersey (39.4°N) and highest male 13

C value found in warmer

waters of the Bahamas (23.3°N) (Figure 2-4A). This gradient could also explain why turtles

foraging in shallow waters off North and South Carolina (35.3 and 33.3°N, respectively) that

were expected to have high 13

C—reflecting benthic feeding—had low 13

C values.

Ultimately, variations in the isotopic signatures by geographical location should also be

reflected in other food web organisms. Isotopic data available in the literature for lower trophic-

level organisms in those geographic locations where male loggerheads migrated reveal that they

follow a similar pattern to that of male loggerheads (Figure 2-6). For instance, known trophic-

level organisms such as omnivorous shrimps and lobsters in the Florida Keys (~ 25°N) show

lower 15

N than those of omnivorous crabs and horseshoe crabs off North Carolina (~ 35°N), and

even similar or lower to those of filter feeding bivalves in Virginia and Delaware (~ 37° and

39°N, respectively). Food-web baseline signatures along the latitudinal gradient used by

loggerheads are scarce; however, nitrogen isotopic signatures of particulate organic matter

(proxy for primary producer) are available for the Florida Keys (~25°N) and coastal waters of

Virginia and Delaware (~ 37° and 39°N, respectively). Nitrogen values range from -0.9 (Macko

et al. 1984) to 3.6‰ in waters of the Florida Keys (Behringer and Butler 2006; Evans et al. 2006;

Lamb and Swart 2007) and 7.2 to 7.7‰ in near-shore waters off Virginia and Delaware

(McKinney et al. 2010). Hence, although I did not assess the isotopic signatures of loggerhead

prey items in all the geographic locations visited by the turtles, the results indicate that the

variation in the 13

C and 15

N of male loggerheads is due to geographic location. Additionally,

because the range of values in males is similar to that of female loggerheads (Figure 2-1b), I

believe that the 2-cluster females probably represent a gradient of North to South geographical

locations used by adult female loggerheads in the NWA. The relationship between female

32

isotopic signatures and geographic areas used are being addressed in another study. My results

highlight the need for knowledge of baseline isotopic signatures when identifying foraging

habitats of highly migratory organisms.

Even though there appears to be a separation of geographic location for at least

northernmost versus southernmost foraging areas (i.e., foraging grounds in the MAB versus

subtropical NWA, respectively) using combined 13

C and 15

N values, isotopic signatures from

the northeast Gulf of Mexico overlap with those of Cape Canaveral (Figure 2-1b). The use of

other markers (such as trace elements) could help reveal unique characteristics of the distinct

geographical locations in the NWA for which carbon and nitrogen are not informative.

Because isotopic signatures of male loggerhead tissues reflect integrated diet and habitat use of

the turtles before their capture, the agreement between isotopic signatures and migration

patternswhich reflects the foraging history after the mating seasonsuggests site fidelity to

foraging areas. Hatase et al. (2002; 2006), McClellan et al. (2010), and Zbinden et al. (2010)

have reported similar agreements between isotopic signatures and migration patterns in female

and juvenile sea turtles. Foraging fidelity in NWA adult female loggerheads has also been

observed through satellite telemetry data (Hawkes et al. 2007, 2011) and has been indicated by

the long-term consistency in SI signatures of scute layers (Vander Zanden et al. 2010).

Relation Between Body Size and 15

N Signatures

A surprising decrease of RBC 15

N with body size was revealed in adult male turtles that

stayed in waters off Cape Canaveral (Figure 2-5). Although the RBC 13

C varied from -15.42 to

-16.91‰, the lack of a relationship between body size and 13

C suggests that males were feeding

on prey utilizing similar carbon sources. Values of 15

N commonly increase with body size due

to diet shift to higher trophic level (Reñones et al. 2002). Among individuals feeding on the same

33

diet, low values of 15

N can provide evidence of a lower nitrogen discrimination value in smaller

juveniles with fast growth rates (Martínez del Rio and Wolf 2005; Reich et al. 2008). Unlike

juveniles, adult turtles grow very slowly after reaching sexual maturity (Bjorndal et al. 1983),

thus no growth effect on 15

N may be evident in adult individuals (Martínez del Rio and Wolf

2005). In our study, smaller adult males may preferentially venture into inshore waters of the

east central Florida where anthropogenic influence has been evidenced in primary producers,

which show elevated 15

N values (Barile 2004). If this pattern is consistent when a larger sample

size is analyzed, the cause for the pattern should be explored.

Implications of the Geographic Variation of Isotopic Signatures

The geographic variation in the isotopic signatures of male loggerheads can potentially

help me understand patterns of migratory connectivity between loggerhead foraging grounds and

breeding areas. In particular, by identifying and differentiating foraging subpopulations within

breeding areas in the NWA, I can assess how breeding populations are structured. This has

important implications in the long term. Changes through time in the relative composition of

individuals from a particular foraging ground observed in a breeding area may provide evidence

of threats to which turtles are exposed (Hatase et al. 2002; Zbinden et al. 2011). For example, in

the NWA, turtles using northern foraging grounds are at a higher risk of sublethal toxic effects

from high concentrations of organic pollutants than are southern foragers (Ragland et al. 2011).

Environmental forces acting on resource availability have been reported to drive the life history

of conspecifics, including leatherback (Dermochelys coriacea) and green (Chelonia mydas) sea

turtles in marine regions (Suryan et al. 2009). Recently, Hatase et al. (2010) found differences in

body size in adult female loggerheads (N = 149) related to differential foraging habitat use.

Similarly, Zbinden et al. (2011) found that nesting loggerheads in the Mediterranean (N = 58)

34

exhibited differences in body size and clutch size associated with geographically separated

foraging areas. In the NWA, however, Hawkes et al. (2007), based on a limited sample size (N

=12), did not find any difference in fecundity measures (clutch frequency, clutch size, body size,

remigration intervals, and inter-nesting intervals) between adult females using northern (N = 9)

versus southern areas (N = 3). In this study, I found no differences in body size between turtles

migrating to cool and highly productive waters of the MAB (Group 3, N = 8) and turtles

migrating south to warm waters of the subtropical NWA (Group 4, N = 3), but my samples size

for southern turtles was small. Further systematic assessment of how differences in nutrient

sources and environmental factors acting on nutrient availability, as well as differences in the

concentration of pollutants, may shape the life history and health of loggerheads is crucial, if we

want to understand changes in loggerhead population abundance in the Atlantic (Witherington et

al. 2009).

Studies using satellite telemetry have shown that female NWA loggerhead turtles follow

different migration patterns (Plotkin and Spotila 2002; Dodd and Byles 2003; Hawkes et al.

2007, 2011; Foley et al. 2008; Turtle Expert Working Group 2009) and show fidelity to foraging

areas with unique environmental characteristics after the mating season (Hawkes et al. 2007,

2011). NWA adult males in this study use foraging grounds (Figure 2-2) similar to those of

NWA females (Hawkes et al. 2011), and the agreement found in my study between SI and

satellite data suggest that males show site fidelity to these foraging areas. The similar patterns in

the use of foraging areas and in the SI values observed between females and males in the NWA,

may indicate that adult males have similar foraging strategiessimilar habitat use, foraging

areas, and movement patternsas those of adult female loggerheads in the NWA.

35

Conclusions

In the present study, adult male loggerheads breeding in Florida revealed a geographic

pattern in the SI values, which indicates that males use isotopically distinct geographical areas

after the mating season. Therefore, SI may help identify foraging subpopulations within a

breeding area and elucidate residency and migration patterns in sea turtles in the NWA.

Moreover, by linking foraging grounds to breeding areas through SI analysis, we can begin to

understand how distinct environmental factors in different foraging grounds affect the biology

and ecology of loggerheads. The agreement between the isotopic signatures and post-mating

movement patterns suggests a foraging site fidelity in male loggerheads that has been observed

in adult females. Also, adult male loggerheads revealed a variation in 13

C and 15

N values

similar to that observed in adult females, suggesting that males and females have similar

foraging strategies. The use of additional markers in combination with isotopic signatures may

help differentiate geographically separated areas with similar isotopic signatures. Understanding

the temporal and spatial distribution of sea turtle populations is essential for the development of

effective conservation and management strategies.

36

Table 2-1. Foraging area, sample size (N), and body size (SCL) of the five groups determined

according to the migration that satellite-tracked male loggerheads followed after the

mating season. SCL: straight carapace length.

Group Foraging area N SCL (cm)

mean ± SD min – max

1 Off Cape Canaveral 10 89.0 ± 2.1 86.6 – 96. 2

2 South Atlantic Bight* 2 88.5 ± 1.5 87.4 – 89.5

3 Mid-Atlantic Bight 8 97.8 ± 5.3 89.0 – 102.8

4 Subtropical Northwest

Atlantic

3 94.1 ± 7.6 86.9 – 102

5 Northeast Gulf of

Mexico

2 98.3 ± 12.4 89.5 – 107.0

* Refers to foraging areas in the South Atlantic Bight not including waters off Cape Canaveral.

37

Table 2-2. Adult male loggerhead population range (maximum – minimum) and variance (Var)

of 13

C and 15

N values in red blood cells (RBC), epidermis (EPI), and plasma (PLA)

samples.

RBC (N = 37) EPI (N = 26) PLA (N = 37)

Range (‰) Var Range (‰) Var Range (‰) Var

13

C 8.15 3.22 7.53 3.93 8.19 3.54

15

N 8.97 5.33 9.68 7.15 8.96 5.39

38

Figure 2-1. Stable isotope ratios (13

C and 15

N) of adult female (N = 310) and male (N = 37)

loggerheads in Florida. (a) Adult female loggerhead signatures show the 2 clusters

identified using 13

C (denoted by open triangles and open circles) by Reich et al.

(2010). (b) Comparison of 13

C and 15

N values of adult female (open symbols) and

male (filled symbols) loggerheads in Florida. Male loggerhead samples were

collected during mating seasons at Cape Canaveral, FL in 2006 and 2007. Female

samples from Florida were collected during nesting seasons in 2003 and 2004.

39

Labels indicate the foraging locations to which satellite-tracked male loggerheads (N

= 25) migrated after the mating season. FL Panhandle refers to the northeast Gulf of

Mexico area in Florida and FL Keys refers to the Florida Keys. Unknown turtles are

males without transmitters and satellite-tracked males for which foraging location

could not be determined. Samples are red blood cells (RBC) for males and epidermis

samples converted to RBC values for females (see Results for regression equation).

Other male samples (epidermis and plasma) show the same pattern as RBC (see

Appendix, Figure A-1).

40

Figure 2-2. Spatial distribution of satellite-tracked male loggerheads from Cape Canaveral,

Florida (filled star) after mating season in April 2006 and 2007. Turtles stayed in

waters near Cape Canaveral (11 - 20; numbered circle) or migrated and remained in

various continental shelf locations (1 through 10 and 21 through 25; numbered

circles). FL Panhandle refers to the northeast Gulf of Mexico area in Florida and FL

Keys refers to the Florida Keys. Dark line denotes the 200 m bathymetry. Dotted

lines separate coastal regions: Mid-Atlantic Bight (MAB) and South Atlantic Bight

(SAB). The Subtropical Northwest Atlantic (SNWA) is also shown. Adapted from

Arendt et al. (2012b).

41

Figure 2-3. Linear relationships between epidermis (EPI, N = 26) and red blood cells (RBC, N =

26) for (a) 13

C and (b) 15

N of adult male loggerheads. The relationship between

these tissues that reflect similar temporal resource assimilation is significant for both

13

C and 15

N (see Results). The solid line is the best-fit line, the dashed lines denote

the 95% confidence interval for the linear regression, and the dotted lines denote the

95% prediction interval (the range in which future observations will fall).

42

Figure 2-4. Stable isotope ratios of carbon (13

C; column A) and nitrogen (15

N; column B) of

red blood cells (RBC; N = 25; open circle), epidermis (EPI; N = 17; open square),

and plasma (PLA; N = 25; open triangle) from adult male loggerheads collected at

Cape Canaveral, FL, versus the latitude to which turtles migrated after the mating

season.

43

Figure 2-5. Red blood cell 15

N versus straight carapace length in male loggerhead turtles that

remained off Cape Canaveral (r2 = 0.64, F1,8 = 15.7, P = 0.004).

44

Figure 2-6. Comparison of stable isotope ratios of 13

C (left) and 15

N (right) of food web

organisms at the different foraging locations visited by male loggerheads after the

mating season (represented by latitude). Mean values are given. For scientific names,

sample sizes, and references see Table 2-3.

45

CHAPTER 3

ASSIGNMENT OF NESTING LOGGERHEAD TURTLES TO THEIR FORAGING AREAS

IN THE NORTHWEST ATLANTIC USING STABLE ISOTOPES2

Background

For endangered migratory fauna, knowledge of demographic parameters is key to

accurately assess the status and trends of populations (Esler 2000; National Research Council

2010; Wallace et al. 2010). Because values of demographic parameters can vary with

environmental conditions at the foraging habitats (Cooch et al. 2001; Chaloupka et al. 2008;

Saba et al. 2008), information on movement patterns and foraging locations of populations are of

crucial importance.

Populations of the loggerhead sea turtle (Caretta caretta) nesting in the Northwest

Atlantic (NWA) represent one of the major nesting aggregations for the species in the world

(Ehrhart et al. 2003). NWA loggerhead nesting aggregations are composed of genetically and

demographically distinct populations (Encalada et al. 1998; Shamblin et al. 2012). Loggerheads

swim hundreds of kilometers from a wide range of foraging grounds to their nesting beaches.

Concern was raised when nesting activity in one of these NWA populations declined markedly

from 1998 to 2007 (Witherington et al. 2009); however, an increase in nesting numbers has been

reported in recent years (Van Houtan and Halley 2011). Anthropogenic threats (Jackson et al.

2001; Witherington et al. 2009; Finkbeiner et al. 2011) and changing oceanographic conditions

(Chaloupka et al. 2008; Saba et al. 2008; Van Houtan and Halley 2011) have been proposed as

the main drivers of fluctuations in sea turtle abundance. Because these factors may change

depending on geographic location (Kot et al. 2010 and references therein), efforts to identify

Reprinted with permission from the authors. Original publication: Pajuelo M, Bjorndal KA, Reich KJ, Vander

Zanden HB, Hawkes LA, Bolten AB (2012b) Assignment of nesting loggerhead turtles to their foraging areas in the

Northwest Atlantic using stable isotopes. Ecosphere 3:art89.

46

foraging grounds of sea turtles are vital to understand spatial and temporal fluctuations in nesting

numbers.

To better understand how environmental changes and human threats at different foraging

grounds affect the various nesting populations in the NWA, it is important to evaluate not only

the demographic parameters of each breeding population, but also the proportions of females in

each breeding population located in different foraging areas. Initial efforts have been undertaken

to understand how differential foraging locations and oceanographic conditions affect

demographic parameters such as clutch size, number of clutches per nesting season, clutch sex

ratio, and female body size in loggerhead populations (Hatase et al. 2002; Hawkes et al. 2007a,

b; Zbinden et al. 2011; Bailey et al. 2012).

Satellite telemetry studies have revealed that NWA adult female loggerheads have at least

two different migration patterns (seasonal shuttling migration and year-round residency) when

they leave the nesting beaches and return to their foraging areas. Females may travel up to

hundreds of kilometers and forage in coastal areas along the U.S. Atlantic coast, Gulf of Mexico,

Cuba, and the Bahamas (Plotkin and Spotila 2002; Dodd and Byles 2003; Foley et al. 2008;

Hawkes et al. 2007a, 2011). They also show site fidelity to their foraging areas, characterized by

different environmental features (Hawkes et al. 2007a, 2011), thus revealing patterns of

migratory connectivity between nesting sites, foraging areas, and wintering areas.

Stable isotope analysis, a technique that uses ratios of stable isotopes of naturally occurring

elements (e.g., carbon, nitrogen), can complement information from satellite telemetry on

population connectivity (Webster et al. 2002). Because stable isotopes in the environment are

incorporated into primary producers and then transferred up the food chain (DeNiro and Epstein

1978; Minagawa and Wada 1984), the isotopic values of tissues of higher trophic level

47

organisms reflect differences in the stable isotope values of primary producers of the

environment in which these organisms foraged (Schell et al. 1989; Minami and Ogi 1997; Burton

and Koch 1999; Kurle and Worthy 2002; Cherel et al. 2007; Pajuelo et al. 2010). These spatial

isotopic differences in primary producers create isotopically distinct regions that can be used to

infer residency and movement patterns of organisms migrating among them (Rubenstein and

Hobson 2004; Graham et al. 2010). Within an organism, different tissues incorporate and

turnover stable isotopes at different rates. In sea turtles, epidermis, keratin, and red blood cells

reflect a longer-term foraging history (Reich et al. 2008). Therefore, such tissues collected for

turtles at breeding areas reflect their dietary history at foraging grounds prior to migration to the

breeding area (Wallace et al. 2006; Caut et al. 2008; Reich et al. 2010; Vander Zanden et al.

2010; Zbinden et al. 2011; Pajuelo et al. 2012; Seminoff et al. 2012).

Stable isotope values of animals can be used to identify their foraging areas if (1)

different foraging areas are isotopically distinct and (2) sampled tissues reflect the isotopic

signatures of the foraging grounds (Rubenstein and Hobson 2004). These requirements are met

for adult loggerheads in the NWA and have been demonstrated for adult males (Pajuelo et al.

2012). Male loggerheads show differences in their stable isotope values reflecting the use of

three geographic areas: the Mid-Atlantic Bight (MAB), the South Atlantic Bight (SAB), and the

subtropical NWA (SNWA) (Pajuelo et al. 2012; Figure 3-1), which represent well-established

biogeographic regions with distinctive biotic and abiotic features (Hutchins 1947; Wilkinson et

al. 2009). Moreover, based on satellite telemetry, adult males appear to use foraging grounds

similar to those of adult females in the NWA (Arendt et al. 2012). The large variation in δ13

C

and δ15

N values from nesting loggerheads in Florida, USA (Reich et al. 2010) is similar to that

48

observed in the satellite-tracked male loggerheads (Figure 3-1), and probably represents a

gradient of north to south foraging locations used by adult female loggerheads in the NWA.

The main objective of my study was to assign loggerhead sea turtles nesting along the

U.S. Atlantic coast to their foraging locations in the NWA using stable isotope analysis. First, I

evaluated whether satellite-tracked adult female loggerheads have the same relationship between

geographic areas and stable isotope values as adult males. Second, I characterized the geographic

areas used by adult loggerheads with isotopic values of satellite-tracked adult loggerheads and

additional turtles with known foraging locations. Then, I compared the isotopic values of nesting

turtles not fitted with satellite transmitters with those of adult loggerheads with known foraging

location to determine their foraging areas based on assignments from discriminant analysis.

Finally, I estimated the proportion of each nesting population foraging in each geographic area.

By combining stable isotope analysis and satellite telemetry to identify foraging locations of

breeding populations, I can then rely on stable isotope analysis alone to assign large numbers of

female loggerheads to their foraging grounds rapidly and at low cost. This knowledge will allow

us to assess with robust sample sizes how different environmental factors and threats at the

different foraging grounds affect the demography of adult loggerheads in the NWA and focus

management and conservation efforts appropriately.

Material and Methods

Data and Sample Collection

Epidermis samples were collected from 87 adult female loggerhead turtles during the

2004 and 2005 nesting seasons (May – Jul) at six nesting areas (Table 3-1; Figure 3-2): Bald

Head Island in North Carolina (BHI; 33.86° N, 77.99° W), and Wassaw (WAS; 31.84° N, 80.98°

W), Blackbeard (BLA; 31.61° N, 81.14° W), Sapelo (SAP; 31.40° N, 81.28° W), Jekyll (JEK;

31.07° N, 81.42° W), and Cumberland (CUM; 30.85° N, 81.45° W) Islands in Georgia.

49

Additionally, 15 adult-size turtles (curved carapace length, CCL ≥ 84 cm) were sampled at one

foraging area in Florida Bay, Florida (24.08° N, 81.03° W) in March and June 2011 (Table 3-1;

Figure 3-2). Previously published isotopic data from epidermis samples of adult female

loggerheads collected at four nesting areas in Florida (N = 310; Reich et al. 2010) and adult male

loggerheads collected at one breeding area in Florida (N = 23; Pajuelo et al. 2012) were also

included in this study (Table 3-1; Figure 3-2).

All epidermis samples were collected using a 6 mm biopsy punch and stored in 70%

ethanol at room temperature until dried at 60C prior to sample preparation and analysis.

Epidermis samples reflect the turtle’s dietary history over a long period of time (i.e., up to 4

months) based on studies conducted on juvenile loggerheads (Reich et al. 2008). An even longer

foraging history is probable in adult loggerheads because rates of isotopic incorporation slow

with reduced growth rates (Reich et al. 2008) and increasing body mass (Carleton and Martínez

del Rio 2005).

Twenty-two female turtles were fitted with satellite transmitters after clutch deposition in

Georgia (N = 18) and North Carolina (N = 4) beaches (Table 1). Hawkes et al. (2007a, 2011)

characterized the distinct movement patterns of these adult female loggerheads and classified

them into two groups, (1) turtles with seasonal migration between summer and winter coastal

areas and (2) turtles with migration to year-round foraging areas. I grouped turtles into three

groups according to the coastal region to where they migrated: the first group is the MAB turtles,

with seasonal migration between summer foraging areas in the MAB and wintering areas in the

SAB; the second and third groups are the SAB and SNWA turtles, with migration to year-round

foraging areas in waters of the SAB and SNWA, respectively. Turtles were tracked for 344.2 ±

148.4 days (mean ± SD) (individuals 5-8, 40-43, 49-52, and 54-63; Hawkes et al. 2011: Table

50

S1). Most of the turtles (N = 19) were tracked for the entire foraging period for turtles using

MAB waters, and for 6 months or more at the foraging ground for turtles using SAB and SNWA

waters. The remaining turtles were tracked for a period of 80 days or more after reaching the

foraging ground. Telemetry data were filtered following Hawkes et al. (2011) to retain location

classes 3, 2, 1 and A, and turning angles greater than 25°, and the latitude of the centroid of the

foraging ground (arithmetic mean of all the filtered location points from the foraging ground)

was used to evaluate the relationship between stable isotope values and geographic location.

Stable isotope values of epidermis samples from satellite-tracked female turtles (N = 22) were

used to characterize the three geographic areas used by adult loggerheads, assuming that isotopic

values from foraging areas used prior to nesting reflected those used post-nesting (identified by

satellite telemetry), as adult female loggerheads are known to exhibit site fidelity to foraging

areas (Hawkes et al. 2007a, 2011; Vander Zanden et al. 2010). Also, because the isotopic values

of satellite-tracked male loggerheads have shown a geographic pattern consistent with the use of

the three geographic areas mentioned above (Pajuelo et al. 2012), male data (N = 23) were

incorporated in the isotopic characterization of the three geographic areas used by adult

loggerheads. Few satellite tracked turtles migrated to the SNWA, so I included additional

epidermis samples from adult-size loggerheads collected at one foraging ground in the SNWA

(Florida Bay; N = 15).

Finally, I used the isotopic values from epidermis samples of nesting turtles not fitted

with satellite transmitters from BHI in North Carolina (N = 18) and WAS in Georgia (N = 47), in

combination with published isotopic data from nesting turtles at four nesting areas in Florida (N

= 310; Reich et al. 2010) (Table 3-1), to compare to those of turtles with known foraging ground

to assign them to one of the three geographic areas used by adult loggerheads in the NWA.

51

Sample Preparation and Analysis

Epidermis samples were washed with deionized water and wiped with isopropyl alcohol

to remove epibionts and extraneous particles. The outermost layer of the turtle epidermis was

separated from the underlying tissue, finely diced with a scalpel blade, and dried at 60C for 24

h. Lipids were extracted from samples with petroleum ether using an ASE300 accelerated

solvent extractor (Dionex).

For stable isotope analysis, 0.5-0.6 mg of each sample was weighed and sealed in a tin

capsule. Samples were analyzed for δ13

C and δ15

N ratios by combustion in a COSTECH ECS

4010 elemental analyzer interfaced via a ConFlo III device to a DeltaPlus XL isotope ratio mass

spectrometer (ThermoFisher Scientific) in the Stable Isotope Geochemistry Lab at the University

of Florida, Gainesville. Results are presented as stable isotope ratios of a sample relative to an

international standard and reported in the conventional notation:

X = [(Rsample/Rstandard) –1] x 1000, where X is the relative abundance of 13

C or 15

N in the

sample expressed in parts per thousand (‰); Rsample and Rstandard are the ratios of heavy to light

isotope (13

C/12

C and 15

N/14

N) in the sample and international standard, respectively. The standard

used for 13

C was Vienna Pee Dee Belemnite and for 15

N was atmospheric N2. The reference

material USGS40 (L-glutamic acid) (N = 22) was used to normalize all results, SD = 0.05‰ and

0.13‰ for 13

C and 15

N, respectively.

Statistical Analyses

The effect of geographic location on 13

C and 15

N values was evaluated with a

Spearman rank correlation test between isotope values and the latitudes of the foraging grounds

of the adult female turtles.

52

To determine the similarity of the isotopic values of samples from turtles of unknown

foraging ground (hereafter referred to as unknown turtles) to those of samples from turtles of

known foraging location (hereafter referred to as known turtles), I classified the isotopic values

of known turtles into three groups: MAB, SAB, and SNWA. Multivariate analysis of variance

(MANOVA) was used to test for variation in 13

C and 15

N values among groups to test if they

were quantitatively discrete. Then, these three isotopically defined groups were combined with

the unknown turtles in a quadratic discriminant analysis, due to unequal variance among groups.

The discriminant analysis assigned each unknown turtle to the geographic area for which it had

the highest probability of membership. To test the accuracy of assignment, I applied the leave-

one-out cross validation method to the reference groups, where a single turtle is removed from

the total and classified to a foraging region by the functions derived from all turtles other than

the excluded turtle, with the process being repeated for each remaining turtle.

Following the determination of geographic area for unknown turtles, I evaluated the

population structure of all breeding populations using only turtles assigned to one of the three

groups with ≥ 80% probability of group membership (Rocque et al. 2006; Seminoff et al. 2012)

(340 out of 375 turtles). Finally, a chi-square test was performed to test for inter-annual variation

in the proportion of turtles using different foraging grounds for nesting beaches that were

sampled in two consecutive years, whenever sample size allowed (i.e., Cape Canaveral-CNS,

Melbourne-MEL, and Juno-JUN beaches; Table 3-1). All data were analyzed using program R

(R Development Core Team 2011) with an level of 0.05.

Results

Epidermis isotopic values of adult females in the NWA ranged from 3.5 to 18.7‰

and -6.9 to -17.6‰ for 15

N and 13

C, respectively (Figure 3-3).

53

The epidermal isotope values from satellite-tracked female loggerheads (tracked long

enough to identify their foraging areas) revealed a geographic pattern: females that migrated

north to seasonal foraging grounds in the MAB (e.g., New Jersey, Virginia, and Delaware) after

the nesting season had high 15

N values and low 13

C values (Figure 3-3). The lowest 15

N value

and highest 13

C value were found in a female that migrated south to a year-round foraging

ground in the SNWA (The Bahamas) (Figure 3-3). Intermediate 15

N and 13

C values were

found in turtles migrating to coastal waters of the SAB (e.g., Georgia and northern Florida)

(Figure 3-3). A significant negative correlation was found between 13

C values and the latitude

to which females migrated after the nesting season (Spearman’s rank correlation rs = -0.64, N =

22, P = 0.001; Figure 4A), while a significant positive correlation was found between 15

N

values and latitude (Spearman’s rank correlation rs = 0.46, N = 22, P = 0.029; Figure 3-4B).

Stable isotope values of epidermis from adult loggerhead males and females using the

same geographic areas were similar. Even though three satellite-tracked turtles had isotopic

values that were not consistent with the geographic area to which they migrated (turtles 1, 2, and

3; Figure 3-5A), I found significant differences in combined 13

C and 15

N values among

geographic areas used by adult loggerheads in the NWA (MANOVA, F = 29.32, P < 0.001).

The isotopic signatures of these three groups were used as a reference from which to compare the

isotopic values of unknown turtles. Discriminant analysis assigned all unknown turtles to one of

the three geographic areas with 91% (340 out 375) of those turtles assigned to a unique

geographic area with a probability ≥ 80% of group membership (Table 3-2; Figure 3-5B). The

percentages of turtles assigned at higher probabilities were lower but remained substantial: 85

and 79% with a probability ≥ 90 and 95%, respectively. Leave-one-out cross validation revealed

a 6% (N = 4) misclassification rate, which corresponded to the misclassification of turtles 1, 2, 3,

54

and an additional turtle. Unknown turtles assigned with ≥ 80% probability to a geographic area

were used to evaluate the foraging structure of breeding populations. A latitudinal trend in the

foraging area use by nesting loggerheads was revealed; the proportion of turtles using the MAB

increased from south to north and the proportion using the SNWA increased from north to south

(Figure 3-6). The majority of turtles (72-80%) nesting at higher latitudes (i.e., BHI and WI) used

foraging areas in the MAB and few turtles (6%) used the SNWA (Figure3-6). Most turtles (46-

81%) nesting at lower latitudes (i.e., Juno Beach and Broward County) used the SNWA and few

(2-21%) used the MAB (Figure 3-6). A large number (36-59%) of nesting turtles from CNS used

the SAB. The use of the SAB declined north and south of CNS (Figure 3-6).

The proportion of turtles using the different foraging areas varied between years in CNS

(Pearson’s chi-square test, df = 2, 2 = 11.51, P = 0.003), MEL (Pearson’s chi-square test, df = 2,

2 =11.39, P = 0.003), and JUN (Pearson’s chi-square test, df = 2,

2 = 22.70, P < 0.001) beaches

(Figure 3-6). A marked pattern in the reduction in the proportion of turtles using MAB waters

and the increase of turtles using SNWA waters was observed in MEL and JUN in 2004 (Figure

3-6).

Discussion

Isotopic Characterization of Geographic Areas Used by Adult Loggerheads in the NWA

In this study, a combination of stable isotope and satellite-telemetry data allowed me to

characterize three main geographic regions used by adult loggerheads in the NWA (Fig 3-5A).

Recent studies have integrated telemetry data to validate marine geographic patterns in 13

C or

15

N values of highly migratory animals such as seabirds (Jaeger et al. 2010) and sea turtles

(Seminoff et al. 2012) over broad spatial scales (e.g., within ocean basins). Here, I present the

55

combined 13

C and 15

N spatial characterization for a highly migratory animal at a regional scale

in the NWA.

Isotopic turnover in epidermis samples of adult loggerhead turtles is estimated to be at

least 4 months (see introduction), longer than the expected migration period between the

foraging area and breeding ground for a satellite-tracked turtle used in this study (~ 1 month,

based on mean travel duration between nesting area and foraging grounds and between foraging

and wintering grounds; Hawkes et al. 2011, L. Hawkes, unpubl. data). Thus, isotopic values of

epidermis tissues from nesting loggerheads should reflect that of the foraging grounds used prior

to migrating to the nesting beaches.

Satellite-tracked nesting loggerheads demonstrated a geographic pattern in the stable

isotope values (Figure 3-3) similar to the one previously observed in satellite-tracked male

loggerhead turtles (Figure 3-1; Pajuelo et al. 2012). Although the MAB turtles use waters of both

the MAB (summer) and SAB (winter), they maintain distinct stable isotope values from those of

turtles that use waters in the SAB year-round. The difference could result from very slow

turnover rates in epidermis of adult turtles. Also, Hawkes et al. (2007a) suggested that seasonal

turtles (i.e., MAB turtles) that migrate south into SAB areas during winter might undergo fasting

during part of the winter. It has been proposed that 15

N values might increase with fasting

duration (Martínez del Rio et al. 2009). Because the extent of the enrichment in 15

N appears to

be tissue-dependent, this hypothesis has received mixed support (Martínez del Rio et al. 2009)

and remains to be tested in sea turtles.

Nesting loggerhead turtles can be assigned to their coastal foraging areas in the NWA

using stable isotope values because NWA adult female turtles show fidelity to their foraging

grounds both within and between years (Hawkes et al. 2007a, 2011), as has been observed in

56

other loggerhead populations (Broderick et al. 2007; Schofield et al. 2010; Thomson et al.

2012). However, some NWA turtles have been found to occasionally use oceanic waters

(Hawkes et al. 2007a, 2011), which may result in unusual stable isotope values. The isotopic

values of three turtles (1, 2, and 3; Figure 3-5A) did not correspond to the geographic area to

which they migrated after the breeding season. The most likely explanation is that these turtles

did not return to the same geographic area from which they originally came. For example, if a

turtle used waters of the SAB prior to its capture in the nesting beach and later migrated to

waters in the MAB, its isotopic values would show lower 15

N and higher 13

C values than

would be expected for a turtle using MAB waters (e.g., turtle 2; Figure 3-5A). Although adult

turtles are generally site-fixed to their foraging grounds, occasional shifts can be expected.

The distinct biotic and abiotic characteristics of the three geographic areas used by

loggerheads—MAB, SAB, and SNWA—likely influence isotopic values of turtles using those

areas. High anthropogenic input appears to raise the 15

N values of primary producers in the

MAB (McKinney et al. 2010). Also, high rates of denitrification, which also raise baseline 15

N

values, have been reported in the MAB (Fennel et al. 2006) although its effect on the 15

N

coastal biota has not been assessed yet (McKinney et al. 2010). In addition, the rate of nitrogen

fixation, which lowers the 15

N values of primary producers, is highest in the SNWA (Montoya

et al. 2002). Available 15

N values of particulate organic matter (a proxy for primary producers)

along the latitudinal gradient used by loggerheads reveal this pattern: nitrogen stable isotope

ratios range from 7.2 to 7.7‰ in near-shore waters off of Virginia and Delaware in the MAB

(McKinney et al. 2010), from 4.0 to 6.4‰ in near-shore waters off of South Carolina and

Georgia in the SAB (M. Pajuelo and M. Arendt unpubl. data), and from -0.9 to 3.6 in Florida

Bay in the SNWA (Macko et al. 1984; Behringer and Butler 2006; Lamb and Swart 2008).

57

Water temperature can also affect 13

C values at the base of the food web by affecting

cell growth rate and dissolved carbonate concentration, which have a direct effect on the 13

C

values of primary producers (MacKenzie et al. 2011). Sea surface temperatures in the MAB

during summer—the season when adult loggerheads mainly use MAB waters—range from 15-

27°C, while water temperatures in the SNWA range from 22.5-28°C year round (Wilkinson et al.

2009). Waters in the SNWA are also characterized by the presence of extensive seagrass

communities (Wilkinson et al. 2009), whose contribution to benthic food webs may be evidenced

by relatively low 13

C values in food web organisms (Fry et al. 1982). Ultimately, variation in

baseline isotopic values will be reflected in higher trophic level organisms such as NWA adult

loggerheads, which prey mainly on benthic invertebrates in coastal waters (Hopkins-Murphy et

al. 2003). Stable isotope values of other food web organisms in the NWA exhibit a pattern

similar to that of adult loggerhead turtles (Pajuelo et al. 2012) and indicate that baseline

differences rather than trophic level differences are driving the large isotopic variation in adult

loggerhead turtles.

While turtles using the MAB and SNWA have distinct stable isotope values (Figure 3-

5A), Pajuelo et al. (2012) found that isotopic values from male turtles using the SAB were

similar to those of turtles using coastal waters in the Gulf of Mexico (which were not included in

our analysis) (Figure 3-1). Because I are interested in determining the foraging locations of

loggerheads nesting along the U.S. Atlantic coast, I need to consider that adult female turtles use

foraging areas in regions other than the NWA. Telemetry studies have revealed that adult female

loggerheads nesting in Florida beaches use coastal waters in both the NWA and the Gulf of

Mexico (Foley et al. 2008). Therefore, the isotopic values reflecting the use of waters in the SAB

for turtles from southern nesting beaches may be confounded with those of turtles using waters

58

in the Gulf of Mexico. Other markers, such as trace elements and lead stable isotopes, may help

differentiate these two foraging areas with similar 13

C and 15

N signatures (M. López-Castro

pers. comm.).

In this study, I chose to define the three loggerhead geographic foraging grounds in the

NWA based on the knowledge that these represent well-established biogeographic regions, each

of which shows distinct oceanographic conditions and faunal communities (Hutchins 1947;

Wilkinson et al. 2009). Because stable isotopes may be influenced by factors (see above) that

vary among the biogeographic areas, I was able to find significant differences in the stable

isotope values of turtles among these three areas. However, within a particular foraging ground,

stable isotope values of loggerheads can vary due to differences in habitat type and/or diet

(Rubenstein and Hobson 2004). Thus, to identify feeding areas at a finer scale than the one

presented here will likely require the use of additional biomarkers (e.g., trace elements).

Foraging Locations of Adult Female Loggerheads in the NWA

For many years, much of what was known about the foraging locations of adult female

loggerheads in the NWA relied on information from flipper tag returns (Bell and Richardson

1978; Meylan et al. 1983; Williams and Frick 2008). While informative, tag return data can be

biased because these data mainly rely on the capture of flipper tagged turtles by fisheries. In

recent years, satellite transmitters have been deployed on nesting loggerheads, which have

provided more accurate information on the post-nesting migratory routes, location of foraging

grounds, and feeding behavior of adult female loggerheads in the NWA (Godley et al. 2008).

However, the expense of satellite tags, which limits the number of individuals that can be

tracked, has prevented more widespread use. Thus, stable isotope analysis, which is low cost and

59

can yield results rapidly, can be useful in identifying foraging areas of a large number of

individuals.

Carbon and nitrogen stable isotopes allowed us to assign most nesting loggerheads to a

distinct geographic area in the NWA at a probability of > 80%. The remaining turtles assigned to

an area with a probability of < 80% do not suggest that they use unidentified foraging locations.

Because I found a latitudinal trend in both 13

C and 15

N along the NWA (Figure 3-4), I believe

that those foraging locations are found within one of the three geographic areas in the NWA. I

could not assign all turtles with a probability > 80% probably as a result of isotopic variation

within each of the three geographic areas that was not captured by the satellite-tracked turtles, or

because they travelled to the Gulf of Mexico.

I found that nesting loggerheads showed geographic segregation of foraging grounds;

northern nesting turtles preferred higher latitude foraging areas while the opposite was seen in

southern nesting turtles. Thus, my initial observations revealed that female loggerheads in the

NWA generally use foraging areas in the vicinity of their natal nesting beaches. These results are

consistent with satellite telemetry data from nesting loggerheads in the NWA. For turtles nesting

in North Carolina, South Carolina, and Georgia, Hawkes et al. (2011) revealed that most females

(N = 48) migrated north to seasonal foraging grounds in the MAB, while few (N = 18) move to

year-round waters of the SAB and SNWA after the nesting season. Similarly, based on smaller

sample sizes, Dodd and Byles (2003) and Foley et al. (2008) revealed that nesting turtles from

southern beaches in Florida migrated to waters in the SNWA and rarely migrated to northern

waters in the MAB.

The use of foraging grounds adjacent to natal nesting areas has been suggested previously

for large juvenile loggerheads in the NWA by mixed stock analysis of mitochondrial DNA

60

haplotypes for aggregations of juveniles along the U. S. east coast (Bowen et al. 2004). The

stable isotope approach used in my study allowed us to sample adult female loggerheads at

various nesting areas— where they are more easily accessible—without having to sample turtles

at the different foraging areas to reveal a similar pattern of foraging ground segregation. Mixed

stock analysis of mitochondrial DNA haplotypes has been widely used to assess the contribution

of various nesting areas to feeding grounds (Bolker et al. 2007). Because this technique relies on

differential haplotype frequencies at the various nesting areas, each nesting individual cannot be

assigned to its foraging ground. The existence of habitat-specific stable isotope signatures allows

stable isotope analysis to assign each individual to its foraging area (Rubenstein et al. 2004).

Thus, geographic assignment models in sea turtles may be improved by incorporating traditional

tools such as genetic analyses, mark-recapture data, and satellite telemetry along with stable

isotope analyses to understand the connection between nesting areas and foraging grounds.

The temporal variation in the proportion of turtles using different geographic areas within

three Florida nesting beaches, CNS, MEL and JUN, suggests differential remigration intervals

may exist (i.e., the period of time between reproductive seasons) among foraging subpopulations.

Given the greater distance that turtles foraging in MAB waters travel to reach southern beaches,

there may be differential remigration intervals for these turtles within a southern nesting

population. Adult female turtles that forage in highly productive waters of the MAB during

summer are known to migrate to the SAB during winter months (Hawkes et al. 2007a). Another

possibility is that turtles using MAB waters seasonally may spend more energy undergoing

seasonal migration (Hawkes et al. 2007a), which may be reflected in longer remigration

intervals. Hawkes et al. (2007a), based on a small sample size of females from a northern nesting

beach in North Carolina, did not find differences in remigration intervals (and other fecundity

61

measures) between females using seasonal foraging areas in the MAB (N = 9) versus year round

areas in the SAB and SNWA (N = 3), suggesting that neither differential foraging/migratory

strategies within this northern breeding population was more advantageous (Hawkes et al.

2007a). However, females using MAB waters in the Hawkes et al. (2007a) study were closer to

their northern nesting beach. Recently, variations in reproductive output and demography due to

inter-basin differences in feeding and movement behavior have been reported in leatherback

turtles (Bailey et al. 2012). Further research is needed to assess whether the pattern observed in

northern nesting loggerheads is consistent with a larger sample size and in different nesting

populations.

My study incorporated previously published stable isotope values of epidermis from adult

female loggerheads nesting in Florida beaches from Reich et al. (2010). Additionally, Vander

Zanden et al. (2010) collected scute (carapace keratin) from a subsample of these loggerheads to

investigate the long-term consistency in resource use through stable isotope analysis of scute

layers. Both studies suggested that large differences in 13

C and 15

N observed could be

accounted for by foraging location (Reich et al. 2010; Vander Zanden et al. 2010). In this study,

I confirmed that stable isotope values of female loggerheads in the NWA reflect their foraging

locations by ground-truthing stable isotope values with information obtained through satellite

telemetry. The much greater number of females that can be assigned to foraging grounds based

on stable isotope analysis than on satellite telemetry will allow robust analyses of foraging

ground effects on demographic parameters such as number of eggs per clutch, number of

clutches deposited during a nesting season, and remigration intervals, which are critical to

understand trends in sea turtle nesting populations (National Research Council 2010).

62

Conservation Implications

In order to effectively manage populations of highly migratory endangered species, an

understanding of spatio-temporal distribution is essential. In the particular case of adult

loggerhead populations in the NWA that use waters over a wide geographic range, knowing

which feeding areas a major nesting population primarily uses is important, because it allows

managers to focus conservation efforts where appropriate.

Adult female loggerhead populations segregate among foraging grounds, which is

promising for refining management strategies. I can identify, at a large scale, what areas are

more or less important for a particular nesting population in the NWA. For example, in this study

I identified that foraging areas in the SNWA are highly important for turtles nesting in Florida

beaches, followed by areas in the SAB; while areas in the MAB are used to a lesser degree.

Fisheries bycatch is one of the major threats for loggerhead turtles in the NWA (Bolten et al.

2010). Research on sea turtle bycatch has revealed spatial and temporal variations in loggerhead

bycatch in U.S. fisheries (Kot et al. 2010; Finkbeiner et al. 2011), with shrimp trawl fisheries in

the SAB, SNWA and Gulf of Mexico accounting for the most interactions with loggerhead

turtles in the U.S. (Finkbeiner et al. 2011). Thus, efforts can be focused in the SAB and SNWA

to assess how fisheries interaction, as well as other environmental factors such as changing

oceanographic conditions and prey distribution, impact fecundity measures of Florida nesting

populations. Additionally, I can further our understanding of how these threats and factors drive

the temporal fluctuations in the proportion of individuals within each nesting population that use

the different foraging areas. However, more northerly and lesser used foraging areas may

currently be important, regarding conservation efforts, because they are used by smaller or more

at-risk nesting populations. These foraging areas may become even more important in the future,

63

if southern turtle populations were to shift northward, as suggested under future climate

scenarios (i.e., if southern beaches become too hot; Hawkes et al. 2007b).

Organic pollutants are another anthropogenic threat to which turtles are exposed in the

NWA (Alava et al. 2011 and references therein). Recent research revealed that adult loggerheads

using northern foraging grounds in the MAB have higher concentrations of organic pollutants

than turtles that use waters off central Florida, and my results support the hypothesis that this

may be due to spatial structuring of foraging grounds by population (Ragland et al. 2011).

Similarly, a recent study found that loggerhead eggs laid in a northern nesting beach in North

Carolina had higher organic concentration of pollutants than eggs laid in southern nesting

beaches in Florida (Alava et al. 2011). These differential threats can also affect demographic

parameters and health of the foraging subpopulations and should also be considered in

management plans.

Conclusions

My study demonstrates that stable isotope analysis can be used parsimoniously to identify

foraging areas of adult loggerheads in the NWA at a regional scale. Future research is needed to

assess if stable isotope analyses, perhaps integrated with other biomarkers such as trace

elements, could identify foraging areas at a finer scale. Additionally, I found that adult female

loggerheads nesting along the U.S. Atlantic coast tend to use foraging areas closer to their natal

nesting beaches; a smaller proportion of individuals undertake migrations to distant foraging

grounds. These results are useful for the design of management strategies for the conservation of

loggerhead turtle populations in the NWA. Assignment of large numbers of nesting females to

foraging grounds with stable isotope analysis will allow future research to explore the effects of

foraging ground location on demographic parameters. The conclusions and methods developed

64

in this study are also relevant for other populations of sea turtles and for other highly migratory

species.

65

Table 3-1. Location (state, breeding/foraging area, and latitude), year of collection, and sample

size of epidermis samples from adult loggerhead turtles with known and unknown

foraging grounds used in this study. State Area Lat (°N) Year n Foraging area Isotope data

source

Breeding area Known§ Unknown

NC Bald Head Island 33.9 2004 10 4 6 This study

2005 12 12 This study

GA Wassaw Island 31.8 2005 47 47 This study

Blackbeard Island 31.6 2005 4 4 This study

Sapelo Island 31.4 2004 2 2 This study

2005 8 8 This study

Jekyll Island 31.1 2004 3 3 This study

Cumberland Island 30.9 2004 1 1 This study

FL Canaveral National

Seashore

28.8 2003 44 44 Reich et al. 2010

2004 31 31 Reich et al. 2010

Melbourne Beach 28.1 2003 60 60 Reich et al. 2010

2004 46 46 Reich et al. 2010

Juno Beach 26.9 2003 41 41 Reich et al. 2010

2004 41 41 Reich et al. 2010

Broward County 26.2 2003 47 47 Reich et al. 2010

Port Canaveral 28.4 2006-07 23 23 Pajuelo et al. 2012

Foraging area

FL Florida Bay 25.0 2011 15 15 This study

Total 435 60 375

Notes: NC: North Carolina, GA: Georgia, FL: Florida; Lat: latitude; n: sample size. §Turtles

fitted with satellite transmitters except for turtles sampled at Florida Bay. NC: North Carolina,

GA: Georgia, FL: Florida; Lat: latitude; n: sample size.

66

Table 3-2. Assignment of adult female loggerheads of unknown foraging location to a

geographic area with ≥ 80% probability of group membership. Values in parentheses

are additional turtles assigned with a probability < 80% of group membership.

Geographic area

State Nesting area Lat (°N) n MAB SAB SNWA

NC Bald Head Island, BHI 33.9 18 10 (2) 3 (2) 1

GA Wassaw Island, WAS 31.8 47 31 (3) 6 (5) 2

FL Canaveral National Seashore,

CNS 28.8 75 10 34 (5) 25 (1)

Melbourne Beach, MEL 28.1 106 13 (2) 37 (2) 50 (2)

Juno Beach, JUN 26.9 82 9 16 (6) 50 (1)

Broward County, BRO 26.2 47 1 14 (3) 28 (1)

Total 375 74 (7) 110 (23) 156 (5)

Notes: NC: North Carolina, GA: Georgia, FL: Florida; Lat: latitude; n: sample size. MAB: Mid-

Atlantic Bight, SAB: South Atlantic Bight, SNWA: Subtropical Northwest Atlantic.

67

Figure 3-1. Distribution of stable isotope ratios (13

C and 15

N) of adult male (N = 25) and

female (N = 310) loggerheads in the Northwest Atlantic (NWA). Male loggerheads

(black symbols) are coded by the foraging area used based on satellite telemetry:

Mid-Atlantic Bight (MAB), South Atlantic Bight (SAB), Subtropical Northwest

Atlantic (SNWA). Two male turtles that used coastal waters of the Gulf of Mexico

(GoM) are also shown. Female data (open circles) are from Reich et al. (2010) and

were sampled at various nesting beaches in Florida; male data are from Pajuelo et al.

(2012a). The right panel depicts the main geographic areas used by adult male

loggerheads in the NWA and the Gulf of Mexico.

68

Figure 3-2. Map showing the locations of the six nesting areas and single foraging ground (filled

symbols) sampled in this study: Bald Head Island (BHI), Wassaw Island (WAS),

Sapelo Island (SAP), Blackbeard Island (BLA), Jekyll Island (JEK), Cumberland

Island (CUM), and Florida Bay (FL Bay). Nesting areas sampled in the Reich et al.

(2010) study (open symbols) are also shown: Canaveral National Seashore (CNS),

Melbourne Beach (MEL), Juno Beach (JUN), and Broward County (BRO).

69

Figure 3-3. Stable isotope ratios (13

C and 15

N) of adult female loggerheads in the Northwest

Atlantic. Females tracked with satellite telemetry (N = 22; black filled symbols) were

sampled during nesting seasons in 2004 and 2005 at Bald Head Island, Sapelo Island,

Blackbeard Island, Jekyll Island, and Cumberland Island, and are grouped based on

the geographic area to which they migrated after the nesting season: Mid-Atlantic

Bight (MAB), South Atlantic Bight (SAB), or Subtropical Northwest Atlantic

(SNWA). Additional females sampled (grey filled circles) in this study and females

sampled in the Reich et al. (2010) study (open circles) of unknown foraging location

are also shown.

70

Figure 3-4. Relationship between (A) carbon (13

C) and (B) nitrogen (15

N) stable isotope values

of adult female loggerheads (N = 22) and the latitude (°N) to which they migrated

after the nesting season. Spearman rank correlation is significant for both 13

C (rs = -

0.64, P = 0.002) and 15

N values (rs = 0.46, P = 0.029). Geographic regions in the

Northwest Atlantic: Mid-Atlantic Bight (MAB), South Atlantic Bight (SAB), and

Subtropical Northwest Atlantic (SNWA) are shown under the x-axis separated by

dotted lines. Dashed lines denote trend lines.

71

Figure 3-5. (A) Stable isotope ratios of carbon (13

C) and nitrogen (15

N) of adult loggerhead

turtles with known foraging location (N = 60) showing three groups representing the

three geographic areas used by adult loggerheads in the Northwest Atlantic: Mid-

Atlantic Bight (MAB) South Atlantic Bight (SAB), and Subtropical Northwest

Atlantic (SNWA).The isotopic values of turtles 1, 2, and 3 fell within a group that did

not correspond to the foraging location as observed through satellite telemetry.

Combined 13

C and 15

N values were significantly different among groups

(MANOVA, F = 29.62, P < 0.001). (B) Stable isotope ratios of carbon (13

C) and

nitrogen (15

N) of 375 adult female loggerhead turtles of unknown foraging ground.

Symbols (same as above) indicate the geographic area to which each individual

unknown female turtle was assigned by the discriminant analysis. Turtles assigned

with a probability ≥ 80% of group membership are shown as filled symbols (N =

340). Open symbols represent additional turtles assigned with a probability < 80% of

group membership (N = 35).

72

Figure 3-6. Breeding population structure according to foraging area used by loggerheads nesting

along the U.S. Atlantic coast as determined through discriminant analysis using

carbon and nitrogen stable isotope values of adult loggerhead turtles with known

foraging grounds as reference data. Foraging areas are Mid-Atlantic Bight (MAB;

white fill), South Atlantic Bight (SAB; grey fill), and the Subtropical Northwest

Atlantic (SNWA; black fill). Nesting turtles from Bald Head Island (BHI) and

Wassaw Island (WAS) were sampled in 2004-2005 and 2005 nesting seasons,

respectively. Florida nesting turtles were sampled in 2003 and 2004 nesting seasons;

results from nesting season 2003 are shown in the main map. Inset shows results

from 2004 for Canaveral National Seashore (CNS), Melbourne (MEL), and Juno

(JUN) beaches. BRO is Broward County beaches. The proportion of turtles using the

different geographic areas varied between years for CNS, MEL and JUN beaches (see

text for statistics). The boundaries of the three geographic areas: MAB, SAB, and

SNWA are depicted by dotted lines.

73

CHAPTER 4

LONG-TERM RESOURCE USE AND FORAGING SPECIALIZATION IN ADULT MALE

AND FEMALE LOGGERHEAD TURTLES

Background

Adult sea turtles migrate from distant foraging areas to breeding areas to mate and

reproduce. Females crawl up onto nesting beaches to lay eggs, but males remain in the marine

environment throughout their lives. As such, encountering male turtles for study is challenging.

Because female sea turtles are easily accessible on the nesting beaches, researchers have

gathered substantial information on their biology and ecology. For instance, studies revealed that

some aggregations of female turtles exhibit variations in foraging strategies (Hatase et al. 2002a;

Hawkes et al. 2006; Zbinden et al. 2011) and show long-term fidelity to their foraging areas

(Broderick et al. 2007; Marcovaldi et al. 2010; Seminoff et al. 2012; Tucker et al. 2014).

A few studies on loggerhead sea turtles, Caretta caretta, have addressed the foraging

behavior and migration patterns of male turtles, and found that male loggerheads appear to use

foraging strategies similar to those of female loggerheads. For instance, male loggerhead turtles

from a population in the North Pacific exhibit the body size related foraging strategy dichotomy,

in which larger individuals forage in coastal waters and smaller turtles use offshore oceanic

waters (Hatase et al. 2002b; Saito et al. 2015), that was previously reported for adult female

loggerheads (Hatase et al. 2002a). In the Northwest Atlantic (NWA), recent studies showed that

male loggerheads are almost exclusively confined to coastal waters (Arendt et al. 2012a,b), just

like adult female loggerheads (Hawkes et al. 2011; Ceriani et al. 2012; Foley et al. 2014; Griffin

et al. 2014), and use foraging areas similar to those of female loggerheads (Arendt et al.

2012a,b). Moreover, a recent study comparing stable isotope values between adult male and

female loggerheads in the NWA suggested that male and female loggerheads use not only similar

foraging areas, but also similar habitats and prey items (Pajuelo et al. 2012b). Thus, loggerhead

74

turtles may not exhibit intra-specific sex-based differences in foraging area use. Additionally, the

foraging site fidelity that was widely reported for females in sea turtle populations was first

reported for an aggregation of male loggerheads in the Mediterranean (Schofield et al. 2010). It

is not certain, however, whether all male loggerhead aggregations exhibit the same pattern of

foraging site fidelity.

Accurate parameter estimates are needed for models of population dynamics to predict

how sea turtles will respond not only to climatic changes, but also to conservation and

management strategies. Most data on sea turtle biology and ecology have focused on results at

the population level, but studies on a wide range of organisms revealed that individual

differences in resource use can strongly influence a population’s ecological and evolutionary

dynamics (see reviews by Bolnick et al. 2003 and Araújo et al. 2011).

Individual specialization in resource use within generalist populations has been widely

reported in natural populations (Bolnick et al. 2003), but only recently have studies quantified

the degree of individual specialization within natural populations (Araújo et al. 2011; Vander

Zanden et al. 2013a; Newsome et al. 2015; Rosenblatt et al. 2015). Among the factors that can

affect the degree of individual specialization are intra-specific competition (Svänback and

Bolnick 2007; Tinker et al. 2008), inter-specific competition (Bolnick et al. 2010), predation

(Peacor and Pfister 2006) and ecological opportunity, i.e. diversity of available resources (Araújo

et al. 2011). Even though ecological opportunity is one of the main conditions for individual

specialization, few studies have assessed how diversity of available resources affects the

magnitude of such specialization among individuals (Herrera et al. 2008; Darimont et al. 2009;

Rosenblatt et al. 2015).

75

Sea turtles record their chronological foraging histories in their scutes, the keratinized

inert tissue covering the carapace. Much like continually growing otoliths in fish (Rooker et al.

2008), baleen in whales (Schell et al. 1989) and the gladius in squids (Lorrain et al. 2012), sea

turtle scutes provide a sequential, long-term record because biomarkers in foraging areas are

incorporated and retained in the inert scute tissue (Vander Zanden et al. 2010). Nitrogen and

carbon stable isotope values, 15

N and 13

C, respectively, have been analyzed in serially sampled

scutes of NWA female loggerhead turtles and revealed that, although generalists at the

population level, nesting turtles exhibit individual specialization in resource use that is

maintained for up to a decade (Vander Zanden et al. 2010, 2013). It is not known whether male

loggerhead turtles exhibit a similar pattern of resource use.

In this study, I had four objectives. I serially sampled scutes of adult male loggerheads to:

1) investigate whether individual patterns of resource use (diet and habitat) within individual

male loggerhead turtles are maintained through time and 2) evaluate the degree of individual

foraging specialization among male loggerhead turtles. Most samples were collected at one

foraging area in South Carolina/Georgia, USA, but a few male and female loggerheads were

sampled at a foraging area in Florida Bay, Florida, USA. I report initial results from these

samples with respect to: 3) the effect of resource availability on the degree of individual

specialization between two loggerhead aggregations that use distinct foraging areas, and 4) inter-

sex differences in resource use in loggerhead turtles.

Methods

Data Collection

Scute samples were collected from 18 male and six female adult-size loggerheads

(straight carapace length, SCL>78 cm) at two foraging areas (Table 4-1). SCL was measured

from the anterior nuchal scute to the posterior notch (Bolten 1999). Samples were taken from the

76

posterior medial region of the third lateral scute, using a 6-mm biopsy punch. Sampling took

place during boreal summers of 2011 through 2013 off South Carolina and Georgia, USA

(SC/GA) in the South Atlantic Bight (SAB), and from March to June 2011 from Florida Bay,

Florida, USA (FLB) in the Subtropical Northwest Atlantic (SNWA) (Figure 4-1). The SAB and

SNWA are well-established biogeographic areas with distinct oceanographic and biological

characteristics (Wilkinson et al. 2009). The three males sampled at FLB in 2011 were also fitted

with satellite transmitters (Wildlife Computers SPLASH10) and their movements were tracked

from 136 to 790 days after release (A. Foley, B. Schroeder, B. Witherington unpubl. data). One

of these turtles had its satellite transmitter replaced in 2013 and was tracked for a total of 851

days. These three turtles remained within the western FLB waters throughout the duration of

tracking and only one made a short excursion to offshore waters.

Scute Preparation and Analysis

Prior to micro-sampling, all scutes were rinsed with deionized water and dried at 60°C

for 24 hr. Each scute sample was glued to a glass slide with the ventral side down and the dorsal

surface (oldest tissue) exposed, and was then micro-sampled in 50-m increments using a

carbide end mill. A previous study determined that each 50-m scute layer, the minimum

amount necessary for stable isotope analysis represents ~0.6 years of resource use in adult

loggerhead turtles (Vander Zanden et al. 2010). In cases for which single layers did not provide

enough material for stable isotope analysis, consecutive 50-m scute layers were combined.

Samples were analyzed for 15

N and 13

C by combustion in a Carlo Erba NA 1500 CNS

elemental analyzer interfaced via a ConFlo II device to a DeltaV Advantage isotope ratio mass

spectrometer in the Stable Isotope Geochemistry Lab at the University of Florida, Gainesville,

USA. Results are presented as stable isotope ratios of a sample relative to an international

77

standard and reported in the conventional notation: X = [(Rsample

/Rstandard

) –1] x 1000, where

X is the relative abundance of 13

C or 15

N in the sample expressed in parts per thousand (‰);

Rsample

and Rstandard

are the ratios of heavy to light isotope (13

C/12

C and 15

N/14

N) in the sample and

international standard, respectively. The standard used for 13

C was Vienna Pee Dee Belemnite

and for 15

N was atmospheric N2. Working standards, L-glutamic acid USGS40 (13

C = -26.39 ‰

and 15

N = -4.52 ‰), and L-glutamic acid USGS41 (13

C = 37.63 ‰ and 15

N = 47.57 ‰)

were used to calibrate results. In addition, a reference laboratory standard, homogenized

loggerhead scute (13

C = -18.36 ‰ and 15

N = 7.68 ‰), was used to examine consistency in

isotopic values in a sample similar to the samples used in this study. The analytical accuracy of

my measurements—calculated as the SD of replicates of standards—was 0.10 and 0.17 ‰ for

13

C and 15

N of L-glutamic acid USGS40 (N = 53), and 0.28 for 13

C and 0.23 for 15

N of L-

glutamic acid USGS41 (N = 10), respectively, and 0.17 and 0.29 ‰ for 13

C and 15

N of scute

standards (N = 27), respectively.

Data Analysis

The total niche width (TNW) of a population is determined by the sum of the within

individual component (WIC), which is the mean variability in resource use within individuals,

and the between individuals component (BIC), which is the variability of resource use among

individuals, such that TNW = WIC + BIC, and the ratio WIC/TNW is used as a measure of the

degree of individual specialization (Bolnick et al. 2003). Values close to 0 indicate that

individuals are specialists or use a narrow range of resources, and values close to 1 indicate that

individuals are generalists or use a wider range of resources (Bolnick et al. 2002). WIC has been

used as a proxy for temporal consistency, as it measures how variable an individual’s resource

use is over time (Matich et al. 2011; Vander Zanden et al. 2013). Thus, following the methods of

78

Matich et al. (2011) and Vander Zanden et al. (2013), I used the variance in 15

N and 13

C,

estimated using the ANOVA framework, to calculate the temporal consistency in resource use

and the degree of individual specialization among male loggerheads. The mean sum of squares

within individuals (MSW) was used as a proxy for WIC:

MSW =∑ ∑ (𝑥𝑖𝑗−𝑥𝑖)

2𝑗𝑖

(𝑁−𝑘)

The mean sum of squares between individuals (MSB) was used as a proxy for BIC:

MSB =∑ ∑ (𝑥𝑖−𝑥)2

𝑗𝑖

(𝑘−1)

where i represents an individual, j represents a single scute layer, N is the total number of

observations, and k is the number of individuals. The sum of MSW and MSB was used as a

proxy for TNW and the degree of specialization was calculated as WIC/TNW.

Body size difference between foraging areas was assessed using a two-tailed t-test, and a

Pearson correlation test was used to evaluate the correlation between thickness of scute and turtle

body size. Comparisons of WIC and WIC/TNW within and between foraging areas were

calculated through non-parametric bootstrapping with 1000 replications. All statistics were

conducted in R (R Development Core Team 2014) with an level of 0.05.

Results

Thickness of scutes ranged from 600 to 1450 m, representing approximately 7.2 to 17.4

years of foraging history (Figure 4-2). The longest records were obtained from scutes of turtles

using the foraging area in the SNWA. However, body size did not differ between turtles sampled

in SC/GA and FLB (t = -0.5431, df = 19.576, P = 0.5932) and scute thickness and body size

were not correlated among all turtles combined (Pearson’s r = 0.068, N = 24, P = 0.752) or

within foraging areas (SC/GA, rs = 0.178, N = 15, P = 0.526, and FLB, rs = -0.345, N = 9, P =

79

0.363) (Figure 4-3). I cannot rule out the possibility that scute growth may be affected by distinct

environmental conditions in the two foraging areas (López-Castro et al. 2014).

Temporal consistency within individuals (as represented by WIC) varied with foraging

area for 15

N, but not for 13

C (Figure 4-4a) for male loggerheads. SC/GA turtles were

significantly more consistent in resource use through time than FLB male turtles (15

N WIC, P <

0.01). Within the FLB foraging area, mean 15

N was significantly higher for males (P = 0.040),

but mean 13

C WIC was not significantly different between males and females (P = 0.346).

Because my male sample size was low in FLB, I combined FLB male and female turtles and

revealed that both mean 15

N and 13

C WIC in combined FLB turtles were significantly higher

(15

N WIC, P = 0.022 and 13

C, P = 0.033) than in SC/GA males, revealing an overall greater

consistency in resource use in SC/GA turtles.

WIC/TNW, representing the degree of individual specialization, had mean values close to

0 (mean WIC/TNW < 0.1, Figure 4-4b), indicating that male loggerheads at both foraging areas

exhibit a high degree of individual specialization. Mean WIC/TNW values were not significantly

different for 15

N (P = 0.193) or 13

C (P = 0.443) among SC/GA and FLB males. However, the

smaller isotopic mean range within individuals compared to that of the population (Table 4-1)

suggests that SC/GA turtles could be more individually specialized. Within a foraging area,

WIC/TNW values were not significantly different between FLB males and females for 15

N (P =

0.649) or 13

C (P = 0.863). Thus, combined FLB male and female turtles exhibited a

significantly lower WIC/TNW for 13

C (P < 0.05) than SC/GA turtles, while WIC/TNW values

were significantly lower for 15

N (P < 0.05) in SC/GA loggerheads than in FLB turtles.

80

Discussion

Temporal Consistency in Resource Use

I found a similar pattern of long-term consistency in resource use in male loggerheads

that has been previously observed in adult female loggerheads from a nesting beach in Florida,

USA (Vander Zanden et al. 2010) and that has been quantified in adult female green turtles,

Chelonia mydas, from a nesting beach in Costa Rica (Vander Zanden et al. 2013a). Unlike the

two previous studies, which sampled turtles at nesting beaches, I sampled adult turtles at their

foraging areas. Thus, I assume that variations in 15

N and 13

C represent dietary and habitat

variations within the foraging area.

Male loggerhead turtles were highly consistent in the use of resources through time in

SC/GA, but not in FLB, in particular with respect to 15

N. Even though my male sample size in

FLB was small, females sampled in this foraging area also exhibited lower temporal consistency

than SC/GA male loggerheads. Temporal consistency in diet and habitat use in SC/GA males is

similar to that of nesting green turtles (WIC < 0.5, Vander Zanden et al. 2013a). FLB males (and

females) were not very consistent in resource use, so that 15

N WIC (>1.0) was higher than that

of oceanic juvenile green turtles (Vander Zanden et al. 2013a), which most likely feed

opportunistically in oceanic waters (Bolten 2003), and were expected to show lower temporal

consistency than adult green turtles (Vander Zanden et al. 2013a). However, comparisons with

other turtle populations, without knowledge of how isotopic values vary at the foraging area,

should be made with caution, because organisms may forage on similar prey items and use

similar habitats, but still exhibit different isotopic values (Cummings et al. 2012). A study that

used both bulk and compound-specific stable isotope analyses showed that the lower SC/GA

loggerhead isotopic variation is caused mainly by base of the food web variation, whereas the

81

greater FLB loggerhead isotopic variation is driven by both baseline variation and dietary

variation among loggerheads (M. Pajuelo et al. unpubl. data).

Loggerhead turtles using waters of the NWA have been shown to exhibit geographic

variation in 15

N and 13

C (Pajuelo et al. 2012b; Ceriani et al. 2014) that can be traced to isotopic

differences at the base of the food web (Pajuelo et al. 2012a). SC/GA male loggerheads exhibited

long-term consistency in 15

N and 13

C, with values in agreement with those found within this

foraging area (Pajuelo et al. 2012a, b). Even though FLB males exhibited a high degree of

isotopic variation within individuals, the overall isotopic values found within (and between)

individuals corresponds to isotopic values expected in turtles using this foraging area (Pajuelo et

al. 2012b; Ceriani et al. 2014; Vander Zanden et al. 2015). These concordant stable isotope

values and the satellite tracks of male loggerheads, which revealed a high fidelity to the west side

of the FLB, indicate that male loggerheads exhibit high foraging-site fidelity.

Foraging site fidelity was also revealed in another aggregation of male loggerheads in the

Mediterranean, using long-term satellite telemetry data (Schofield et al. 2010). Thus, foraging

site fidelity may be characteristic of male loggerhead turtles in general. Consistent use of a

known foraging ground that provides sufficient resources is considered a more beneficial

strategy than wandering through unexplored new areas (Schofield et al. 2010). However, this

behavior can prove detrimental if foraging areas become heavily impacted by anthropogenic

activities. A recent study showed that loggerhead turtles in the Gulf of Mexico continued to

forage near the site of the disastrous 2010 oil spill, thus risking exposure to the effects of oil and

chemical dispersants (Vander Zanden et al. submitted).

The low isotopic variation within SC/GA male loggerheads indicates that turtles may be

consistently feeding on similar prey items or on prey items with similar isotopic composition.

82

Stomach content analysis in loggerheads at this foraging area has revealed that turtles rely on

prey items with similar trophic level such as crabs and that some turtles were selectively

consuming particular prey items (Youngkin 2001). Only one SC/GA male loggerhead showed a

large decrease in both 15

N and 13

C values, followed by a return to isotopic values similar to

those before the decrease (Figure 4-2). These values, however, were within the isotopic values

expected for turtles within this foraging area. Such great changes in both 15

N and 13

C values

suggest that the turtle moved to another area within its foraging area where both isotope values

were different at the base of the food web. It could also indicate that when the turtle moved to a

new habitat (13

C), it utilized prey items of lower trophic position, which would have lowered

the 15

N values.

FLB is located in the SNWA, whose warm waters are characterized by great biotic and

habitat diversity. Benthic invertebrate diversity along the US east coast is greater in the FLB

(Roy et al. 1998) as are the available habitats such as seagrass beds, mangroves, and coral reefs

(Wilkinson et al. 2009). And because isotopic variations in FLB loggerheads are also linked to

dietary variations (M. Pajuelo et al. unpubl. data), the larger 15

N variation observed within male

loggerheads using FLB waters suggests that these turtles exploit a variety of resources with

isotopic values that are different from those of turtles using SC/GA waters. However, visual

inspection of Figure 4-2a reveals shorter intervals with less isotopic variation within FLB turtles.

Thus, FLB turtles appear to be consistent in the use of resources for shorter periods of time.

Although there was great variation in 15

N values of FLB males, there was not a

corresponding variation in 13

C. Consistency in 13

C values was similar for male loggerheads at

both foraging areas. However, when combining FLB male and females (justified because WIC

values for both 15

N and 13

C are not significantly different) and comparing them to SC/GA

83

males, I find that SC/GA turtles are also more consistent in 13

C. This is not surprising, as carbon

sources (e.g., seagrass, macroalgae, and phytoplankton) in the FLB have wide-ranging 13

C

values, from -18‰ to -6‰ (Behringer and Butler 2006), which suggests that FLB turtles may

consume prey items that use different carbon sources. Overall, loggerhead turtles show 13

C

values over time that are strikingly more consistent than their 15

N values. This indicates that

turtles are consistently using the same foraging habitat (reflected by 13

C), while the amount of

dietary variation (reflected by 15

N) may depend on the availability of resources within a

particular foraging area.

Individual Specialization

Exhibiting temporal consistency in resource use alone does not necessarily indicate the

degree of individual foraging specialization in a population, as the latter will depend upon how

individuals are partitioning the range of available resources (Bolnick 2003). However, it is

essential to obtain information on individual resource use through time to understand whether the

pattern of resource use (i.e., individualization or generalization) is maintained over time.

In this study, SC/GA male loggerheads exhibited both consistency in resource use

through time and a high degree of individual specialization, similar to those observed in nesting

loggerheads (Vander Zanden et al. 2010) and green turtles (Vander Zanden et al. 2013a). The

isotopic range within male turtles is smaller than the total isotopic range found across all

individuals at each foraging area, revealing that male turtles are part of a generalist population

with specialized individuals. However, Vander Zanden et al. (2010, 2013a) reported on

individual specialization in a nesting population, which was composed of individuals that used

different foraging areas. Because geographic area can account for the large isotopic variation

found in nesting sea turtles (Pajuelo et al. 2012a, b; Vander Zanden et al. 2013b), I cannot

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compare the degree of individual specialization found in nesting turtles to that of turtles from one

foraging ground.

The first observations of dietary specialization in long-assumed opportunistic loggerhead

turtles, were reported by Ruckdeschel and Shoop (1998). They analyzed stomach contents of

hundreds of loggerhead turtles and showed that some individuals selectively consumed the same

prey types. However, the dietary information obtained from stomach contents reveals what an

organism consumed within the past few days, not whether the organism’s dietary preferences are

maintained through time. Even though I do not have information on the specific prey items or

groups of prey items the SC/GA male loggerheads consume in this foraging area, the isotopic

evidence reveals that males forage consistently on the same prey items or groups of prey items

with similar isotopic values over a long period of time. This also suggests that availability of

resources in SC/GA waters remained consistent or that environmental variability that affects

resource abundance was low, so that turtles could maintain individualized foraging behaviors for

long periods of time.

Male individuals in SC/GA waters appeared to be more specialized in 15

N than FLB

loggerheads males (both males and females), whereas FLB turtles were more specialized in 13

C,

suggesting that foraging area may also affect the magnitude of specialization exhibited by

individuals. The lower individual specialization for 15

N found among FLB turtles could be a

consequence of the larger variation in individual resource use over time and that this variation in

resource use, for some individuals, almost encompasses all available resources in the foraging

area. Although FLB individuals are more variable in their use of 13

C than SC/GA turtles, FLB

turtles still appear to be using a narrower range of carbon sources than what is available. Thus,

while a wider range of resources (as reflected by the wider isotopic variation in both 13

C and

85

15

N) is available for FLB turtles, they appear to be more specialized in their habitat use

(reflected by 13

C) than in their dietary preferences (reflected by 15

N). However, some FLB

individuals appear to specialize their diet and habitat use for shorter periods of time, as

evidenced by shorter intervals with less isotopic variation within FLB turtles. Thus, even though

individual loggerheads in general do specialize their diet and habitat use, this behavior is not

consistent in the long term for turtles in FLB waters. These results differ from those of previous

studies in which an increase in resource diversity increased individual specialization. Darimont

et al. (2009) found that insular populations of wolves (Canis lupus) had higher among-individual

dietary variation than wolves inhabiting mainland areas. Island wolves are apparently exposed to

more diverse food sources and habitats (terrestrial and marine) than mainland wolves, which rely

on terrestrial prey items in a more homogeneous habitat (Darimont et al. 2009). Similarly,

Rosenblatt et al. (2015) revealed that American alligators (Alligator mississippiensis) exhibited

higher individual specialization in coastal areas, where alligators had access to a wider variety of

aquatic habitats and prey items, than in freshwater lakes. Common in both studies was the

expansion of the population niche width (TNW), a consequence of the increase in available

resources, which affects individual specialization (WIC/TNW) (Bolnick et al. 2003). Population

niche width also increased with resource diversity in loggerhead turtles (this study). However,

unlike the above studies that based results on short-term data, analysis of isotope values in turtle

scutes (an archival tissue) enabled me to examine the degree of individual foraging specialization

over a longer period of time (~7 to 17 years).

Why are FLB loggerhead turtles less consistent in their foraging behavior over a long

period of time? FLB is a diverse and highly dynamic ecosystem (Fourqurean and Robblee 1999).

86

FLB has experienced dramatic ecological changes since the late 1980s, including seagrass die-

offs and phytoplankton and cyanobacteria blooms, which have had repercussions on the diversity

and abundance of food web organisms such as fish, crustaceans and sponges (Fourqurean and

Robblee 1999; Matheson Jr. et al. 1999; Peterson et al. 2006). Given that loggerhead turtles are

considered generalist carnivores (Hopkins-Murphy et al. 2003), I hypothesize that changes in

abundance of benthic invertebrates in the FLB may have led loggerhead turtles to diverge from

their preferred diet items. Preliminary data from FLB loggerhead gut contents and feces reveal

that the turtles often rely on sponges (B. Stacy pers. comm. and B. Witherington unpubl. data).

Large sponge die-offs within the FLB have been attributed to persistent cyanobacteria blooms

(Peterson et al. 2006). Thus, it is likely that those turtles that rely on sponges may switch to other

available prey items when sponges are not available. A similar diet switch was found in a coastal

loggerhead population, where a long-term study of stomach contents revealed that the diet of the

turtles shifted from benthic invertebrates to fish over the course of 20 years because of declines

in commercially important invertebrate prey (blue crabs, Callinectes sapidus, and horseshoe

crabs, Limulus polyphemus, Seney and Musick 2007). Thus, in the short-term, FLB turtles may

exhibit a preference for a particular prey type, but their diet may change over a long period of

time (more than a decade) because of environmental change.

Intra- and inter-specific competition may also influence foraging decisions of individuals

over time. Experimental and observational studies have found that high intra-specific

competition leads to increased individual specialization (Araújo et al. 2011), whereas inter-

specific competition weakens individual specialization (Bolnick et al. 2010). Data on loggerhead

population density, prey abundance and possible competitors needed to assess intra-specific and

87

inter-specific competition, are not available for each foraging area and thus, I cannot address

these topics at the moment.

My initial results showed a sex-based difference in temporal consistency of resource use,

but not in the degree of individual specialization among FLB loggerhead turtles. These results

suggest that individual female loggerhead diets are more restricted than those of males. Male and

female individuals within a population face different evolutionary and energetic pressures, in

particular related to reproduction (Elliot Smith et al. 2015). Thus, sex-based differences in

resource use are not uncommon. Because my FLB male sample size was small, the differences in

individual specialization between sexes should be interpreted with caution. More studies to

replicate the extensive work conducted on female sea turtle movement patterns and feeding

behavior will allow us to evaluate any sex-based differences in resource use.

Conclusions

My study shows that SC/GA male loggerheads exhibit consistency in resource use over

time and a high degree of specialization of resource use similar to that reported for female

loggerhead and green turtles. The long-term consistency in resource use found among SC/GA

male loggerheads revealed that, similar to female sea turtles, males also exhibit fidelity to their

foraging areas. This long-term consistency also suggests that resource abundance may have been

consistent in waters of the SC/GA, leading to a consistency in male loggerhead foraging

behavior. The initial results reported here on population-level variation in temporal consistency

and individual specialization in resource use among loggerhead turtles that use distinct foraging

areas, provide insights into how resource diversity and abundance may affect the foraging

behavior of loggerheads. Moreover, initial results showed sex-based differences in individual

specialization, suggesting that males may exploit a greater range of resources than females.

Future research should examine this pattern with larger sample sizes and in other populations to

88

test the validity of my results. As higher trophic level organisms, loggerhead sea turtles play an

important role in their ecosystems. Understanding how sea turtles utilize resources will allow us

to predict how they will respond and whether they will be able to adapt to changing climate and

environmental conditions.

89

Table 4-1. Mean and range of carapace length for individual loggerhead turtles, and mean and

total range of 15N and 13C values of loggerhead turtle scute tissue sampled at two

foraging areas, South Carolina/Georgia (SC/GA) and Florida Bay (FLB). For isotope

values, mean is the mean range of isotopic values within individual turtles and total is

the range of isotopic values across all individuals. Foraging area Sex N Layers SCL mean, range (cm)

15N mean, total (‰)

13C mean, total (‰)

SC/GA M 15 6 – 21 83.5, 10.9 1.55, 4.59 1.47, 4.08

FLB M 3 12 – 22 84.2, 2.4 3.40, 6.85 3.06, 4.69

F 6 18 – 29 84.4, 9.0 3.72, 6.42 2.42, 6.01

N is number of individuals, Layers indicate the range of scute samples analyzed per turtle at each foraging area and

per sex, and SCL is straight carapace length.

90

Figure 4-1. Foraging locations, South Carolina/Georgia and Florida Bay, where male

loggerheads turtles were sampled for scute tissue. Foraging areas are found within

distinct biogeographic areas (delimited with dotted lines): South Atlantic Bight (SAB)

and Subtropical Northwest Atlantic (SNWA).

91

Figure 4-2. Resource use of individual male loggerhead turtles (N = 18) as indicated by (a) 15

N

and (b) 13

C values of successive scute layers. Foraging areas are South

Carolina/Georgia (SC/GA) and Florida Bay (FLB). Arrow indicates the FLB male

loggerhead that made a short excursion to oceanic waters. Resource use of individual

female loggerheads (N = 6, dashed lines) is also included for comparison in the FLB.

92

Figure 4-3. Thickness of scute in relation to straight carapace length in loggerhead turtles from

South Carolina/Georgia (open circles) and Florida Bay (filled circles).

93

Figure 4-4. Comparison of (a) temporal consistency (WIC) and (b) degree of individual

specialization (WIC/TNW) between loggerhead turtles from two foraging areas,

South Carolina/Georgia (SC/GA) and Florida Bay (FLB). Circles represent mean and

error bars represent the standard error. Results for SC/GA are for male turtles only

while both male and females are shown for FLB. Groups that do not share letters in

common are significantly different ( = 0.05).

94

CHAPTER 5

SUMMARY AND FUTURE RESEARCH

Summary

The aim of this study was to further the understanding of the foraging ecology of

loggerhead turtles in the Northwest Atlantic using stable isotope analysis. Results show that on a

broad scale, highly elusive male loggerhead turtles exhibit similar foraging strategies to those of

widely studied female loggerheads, as male stable isotope values indicate they feed on similar

prey items and use similar foraging areas as females (Chapter 2). At a more regional scale,

results show that male loggerheads also exhibit individual foraging specialization that is

maintained for up to 17 years (Chapter 4). Consistency in temporal resource use, as reflected in

stable isotope values, suggests that male loggerheads consistently forage on similar prey items or

groups of prey items and use the same foraging habitats through time. These results reaffirm that

although loggerhead turtles are generalists at the population level, individual turtles partition the

resources available at their foraging areas and become specialists at the individual level. Previous

observations from stomach content analysis showed that some loggerhead turtles selectively

consumed particular prey or groups of prey items (Ruckdeschel and Shoop 1998). However, by

using scute samples (an archival tissue) from male loggerheads, it was possible to obtain both

dietary and location information over many years, which shows that male turtles also exhibit

specialized foraging behavior, previously reported only in female sea turtles.

Initial results of the effect of resource availability on the degree of individual

specialization among loggerhead aggregations suggest that loggerheads that use areas with a

greater diversity of resources may exhibit a lower degree of individual resource specialization

(Chapter 4). This is not in agreement with recent findings that high diversity of resources

increases the degree of individualization (Rosenblatt et al. 2015). I suggest that the degree of

95

individual specialization is context-dependent. If a foraging area with high diversity of resources

experiences changing environmental conditions, then individual turtles will be able to specialize,

but for shorter periods of time compared to turtles using more stable, yet less diverse

environments. Limited environmental change in a foraging area will allow individuals to

specialize on reliable food sources over long periods of time.

By combining stable isotope data with satellite telemetry data, I was able to validate the

use of stable isotope data alone to assign loggerhead turtles to their foraging areas in the NWA

(Chapter 3). I successfully used stable isotope data to identify foraging areas of loggerheads,

because the different areas studied were isotopically distinct. The foraging areas used by

loggerheads in the NWA are found within three major biogeographic regions, whose distinct

biogeochemical processes set the characteristic isotopic values found in particulate organic

matter (a proxy for primary producers), which were also observed in loggerhead turtles. Previous

work reported a wide range of isotopic values in nesting loggerhead turtles, but researchers were

not able to distinguish between location or diet as the cause of the wide range in isotopic values

(Reich et al. 2008; Vander Zanden et al. 2010). This study shows that the large isotopic variation

found among loggerheads is mainly driven by location differences.

The stable isotope technique developed in this study allowed the assignment of more than

300 nesting turtles from five nesting beaches along the U.S. east coast to their foraging areas,

and found that nesting loggerhead turtles tend to use foraging areas closer to their natal nesting

beaches. Additionally, initial findings revealed temporal variation in the contribution of foraging

areas to various nesting beach populations, suggesting differences in remigration intervals among

turtles with different foraging area origins. A later study that expanded on these results found

similar results in a northern loggerhead nesting aggregation, in which the proportion of turtles

96

originating from different foraging areas varied over seven years, indicating that the recent

increase in nesting population numbers had been driven by an increased contribution of turtles

from one foraging area in particular (Vander Zanden et al. 2014). Thus, the technique developed

in the present study (Chapter 3) allowed for the monitoring of the inter-annual variability in

nesting population abundance (Vander Zanden et al. 2014).

In summary, this study expanded our knowledge of loggerhead foraging strategies and

demonstrated that 13

C and 15

N of loggerhead turtles are effective biochemical tags to link

loggerhead foraging grounds and breeding areas.

Significance and Implications

Findings of this study have several implications for the use of stable isotopes in marine

environments and for the conservation and management of loggerhead turtles. First, I reaffirmed

the importance of knowledge of baseline isotopic variation when comparing the resource use of

populations using distant foraging areas. Thus, future studies should take into account the

isotopic values at the base of the food web when interpreting stable isotope data. The use of

compound-specific stable isotope analysis of amino acids (CSIA-AA), which has expanded in

recent years, allows effective differentiation between trophic/dietary effects and baseline isotopic

shifts (Popp et al. 2007). Thus, CSIA-AA should be incorporated in sea turtle research using

stable isotopes for dietary evaluation and to identify baseline isotopic values.

The wide range of foraging areas that the loggerhead turtles use in the NWA exposes

them to a variety of distinct environmental conditions and anthropogenic threats, which have

been proposed as drivers of loggerhead nesting population numbers (Witherington et al. 2009;

Arendt et al. 2013). Thus, identifying major loggerhead foraging areas is key to understanding

spatial and temporal fluctuations in nesting population numbers. This study relied on both

97

satellite telemetry and stable isotopes to link regional foraging grounds to breeding areas, which

will enable assessment of how anthropogenic threats such as fisheries interactions affect nesting

population numbers. This approach can also be useful to determine the origin of stranded turtles,

as it can reveal whether turtles were residents or migrants to the area where they were stranded,

and assess unusual stranding/mortality events.

The high degree of individual foraging specialization found in both male and female

loggerheads reveals that loggerheads in general are able to partition a wide range of available

resources. This foraging specialization can be maintained for many years, but can also change,

possibly in response to unfavorable environmental conditions that affect prey

distribution/abundance. If indeed loggerhead populations are able to exploit resources, even

under changing environmental conditions, it could imply that they will have greater potential to

respond to climate change.

The long-term consistency in resource use indicated that male loggerheads also exhibit

site fidelity to their foraging areas. This behavior can prove harmful for loggerhead populations

that are exposed to anthropogenic threats. For instance, Vander Zanden et al. (submitted)

revealed that loggerheads in the Gulf of Mexico continued to use areas that were impacted by the

2010 oil spill and were exposed to harmful oil contaminants and chemical dispersants used in

cleanup efforts.

Future Research

The stable isotope approach developed in this study allows the identification of foraging

areas at the regional level in the NWA, as turtles using the same biogeographic area shared

similar isotopic values. However, some turtles that use waters in the Gulf of Mexico had isotopic

values similar to turtles in one area of the NWA. The use of stable isotope values, in combination

with other biochemical tags, could allow for finer-scale identification of loggerhead foraging

98

areas in the NWA and help differentiate geographically separated areas with similar isotopic

values. Trace elements, in combination with stable isotopes, have been used successfully to

determine the origin of fish to estuaries along the US east coast (Anstead et al. 2015). Trace

elements have been analyzed in scute of sea turtles to link the contribution of oceanic habitats to

neritic foraging grounds (López-Castro et al. 2013) at a very large scale. Future studies should

implement this approach in the NWA and Gulf of Mexico, where the presence of estuaries that

are known to influence coastal waters should present distinct trace element ratios that can be

reflected in the loggerheads scute tissue. A finer-scale resolution in the identification of sea turtle

foraging areas could allow managers to focus efforts on areas of high conservation value. Thus,

the stable isotope approach implemented in this study is valuable, because it allows identification

of the foraging area of large number of turtles at a low cost, but it can still be refined.

Another approach to improving the assignment of sea turtles to their foraging areas was

validated by Vander Zanden et al. (2015). A continuous surface of stable isotope values was

created to assign the foraging origin of nesting loggerheads in the Gulf of Mexico. Because

loggerheads from multiple areas in the Gulf of Mexico had similar isotopic values, it was

difficult to assign turtles to their foraging areas accurately. However, the advantages of this

approach are that all individuals can be assigned to the continuous surface and individual results

can be aggregated to identify hotspots of foraging area use (Vander Zanden et al. 2015). This

approach can be implemented in the NWA to identify the foraging areas of nesting populations

that are known to use the NWA almost exclusively.

Because of the opportunistic nature of loggerhead turtles that can specialize on a wide

range of resources, as I have reaffirmed in this study, I caution against the use of bulk stable

isotope analyses alone to address trophic structure questions. CSIA-AA has proven useful to

99

obtain information from both the base of the food web and the trophic level of the consumer.

However, this approach has still not been validated in sea turtles. Only relative trophic level

estimations have been obtained for sea turtles because discrimination factors have not been

obtained for any sea turtle species, which prevents trophic comparisons between sea turtles and

other species using the same foraging grounds.

Initial results evaluating the sex-based differences in loggerhead turtles suggest that

female loggerheads are more restricted in resource use than are males. Further studies with larger

sample sizes will allow me to evaluate any sex-based differences in resource use. Future studies

should address how varying degrees of individual variation in resource use within females can

have demographic consequences on the population.

The recent increase in loggerhead numbers poses new challenges. I do not know what the

effect of increased numbers of loggerheads will be on their ecosystems. What will the effect be

on the relationship between loggerheads and ecologically similar species? For instance, in the

NWA, Kemp’s ridleys, Lepidochelys kempii, are found within the same coastal foraging areas as

loggerheads and their diet may include similar prey items as those of loggerhead turtles (Seney

2002; Witzell and Schmid 2005). However, the degree of ecological interaction between these

two sea turtle species has not yet been assessed. Future studies should evaluate how these two

species partition resources to further our understanding of how these two species coexist in the

same environment.

100

APPENDIX

EPIDERMIS AND PLASMA ISOTOPIC VALUES

Figure A-1. Stable isotope ratios (13

C and 15

N) of epidermis (EPI; N = 26; a) and plasma

(PLA; N = 37; b) samples of adult male loggerheads in Florida. Samples were

collected during mating season at Cape Canaveral, FL. EPI and PLA values show the

same pattern as the one observed in red blood cells (see Figure 2-1b). Labels indicate

the geographic location to which satellite-tracked males migrated after the mating

season. Unknown turtles are males without transmitters and satellite-tracked males

for which foraging area could not be determined.

101

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BIOGRAPHICAL SKETCH

Mariela E. Pajuelo was born in Lima, Perú. She attended María de la Providencia School,

where she graduated in 1993. She then attended Universidad Nacional Mayor de San Marcos in

Lima, Perú where she graduated in 2001 with a focus in Hydrobiology and Fisheries Science. In

2004, she joined Peruvian non-govermental organization Pro Delphinus to work as a research

associate conducting work on conservation of marine mega-fauna. In fall 2007, she started

master´s studies at the University of Florida (UF) with Dr. Karen A. Bjorndal, where she focused

on the trophic ecology of oceanic juvenile loggerhead sea turtles. In fall 2010, she began doctoral

work under guidance of her former master’s advisor, studying the foraging ecology of

loggerhead turtles in the North Atlantic Ocean. Mariela graduated in the fall of 2015 with a

degree in zoology.