1 STABLE ISOTOPE DICHOTOMY IN LOGGERHEAD TURTLES REVEALS PACIFICATLANTIC OCEANOGRAPHIC DIFFERENCES By MARIELA E. PAJUELO A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2010
2 2010 Mariela E. Pajuelo
3 To my parents, Fortunato and Ricardina
4 ACKNOWLEDGMENTS I would like to express my gratitude and appreciation to all of those who contributed to making my project possible one way or another. So, here it goes. First, I especially thank Dr. Jeffrey A. Seminoff who introduced me to a fascinating technique: Stable Isotope Analysis and encouraged me to develop this project. At Pro Delphinus in Peru, I would like to thank Joanna Alfaro-Shigueto for giving me the opportunity to get involved in marine conservation and everything this entails in a country such as Peru, Jeffrey C. Mangel for proof-reading and commenting everything I wrote, and Celia Cceres Bueno for the invaluable help during field work. Collection of samples would not have been possible without the enthusiastic help of fishermen Francisco Chaval Bernedo, Mateo Mamani, and Miguel Cuentas. Pro Delphinus team is also responsible for making work in the least desirable conditions fun. In Peru, I also thank IMARPE-Ilo for letting me use their facilities to process samples. My lab members Kim Reich, Hannah Vander Zanden, and Melania Lpez-Castro, were all incredibly great chicas and helped in various aspects of my research. Other friends at the department Carlos Manchego, Patty Zrate, Jordan Smith and Elvis Nuez are thanked for making my time here fun. I warmly remember my friends outside the department, Jorge Lingan, Andrea Pino, and Oriol Pealver for their friendship and the many good laughs (and e-mails) shared during the time I spent working on my research project. My parents Ricardina and Fortunato and siblings Eli, Ethel and Edgar deserve a lot of gratitude for their continued moral support, encouragement, and unconditional love. I also appreciate the support Lucas Majure has given me, and I love him very much because of it.
5 And most of all, I would like to thank my advisor, Dr. Karen A. Bjorndal, and Dr. Alan B. Bolten for their patience, guidance, invaluable knowledge provided, and encouragement throughout these years. It was their vision, along with that of Dr. Seminoff, that led my project to its completion. Last, I would like to acknowledge the support of Fulbright Scholarship (Peru), Dexter Fellowship in Tropical Conservation Biology, Lerner-Gray Grants for Marine Research at the American Museum of Natural History, and Brian Riewald Memorial Fund at the University of Florida for grants awarded to conduct my research.
6 TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................. 4! LIST OF TABLES ............................................................................................................ 7! LIST OF FIGURES .......................................................................................................... 8! ABSTRACT ...................................................................................................................... 9 CHAPTER 1 INTRODUCTION..................................................................................................... 11! 2 MATERIALS AND METHODS ................................................................................ 17! Data and Sample Collection .................................................................................... 17! Sample Preparation and Analysis ........................................................................... 18! Statistical Analysis ................................................................................................... 20! 3 RESULTS ................................................................................................................ 23! 4 DISCUSSION .......................................................................................................... 30! Differences between Ocean Regions ...................................................................... 30! Interannual Variation of Loggerhead !13C and !15N within O cean Regions ............ 36! Relationship between !15N and Turtle Size ............................................................. 39! 5 CONCLUSIONS ...................................................................................................... 42! LIST OF REFERENCES ................................................................................................ 43! BIOGRAPHICAL SKETCH ............................................................................................ 51!
7 LIST OF TABLES Table page 3-1 Stable isotope ratios (!15N and !13C) of marine organisms in the southeast Pacific and the northeast Atlantic. ....................................................................... 26!
8 LIST OF FIGURES Figure page 1-1 Schematic !15N signatures in the upper water column in the presence of nitrogen fixation and denitrification.. ................................................................... 16! 2-1 Collection sites of samples off southern Peru in the southeast Pacific. .............. 21! 2-2 Collection sites samples in Azorean waters in the northeast Atlantic. ................ 22! 3-1 Stable isotope ratios (!15N and !13C) of skin samples from juvenile loggerheads Caretta caretta in the southeast Pacific and in the northeast Atlantic ................................................................................................................ 27! 3-2 Comparison of stable isotopes ratios of the food web components in the southeast Pacific and in the northeast Atlantic. .................................................. 28! 3-3 Skin !15N vs. curved carapace length in loggerhead sea turtles Caretta caretta in the southeast Pacific and in the northeast Atlantic. ............................ 29!
9 Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Master of Science STABLE ISOTOPE DICHOTOMY IN LOGGERHEAD TURTLES REVEALS PACIFICATLANTIC OCEANOGRAPHIC DIFFERENCES, By Mariela E. Pajuelo May 2010 Chair: Karen A. Bjorndal Major: Zoology Denitrification and nitrogen fixation processes in the marine environment have been intensively studied, particularly how these processes affect the delta^15 N signature of inorganic nutrients and organisms at the base of the food web. The assumption that these delta^15N differences at the base of food webs are reflected in higher trophic level organisms, however, has been neglected. In this study, I evaluated whether an ocean basin delta^15N dichotomy was evident in oceanic juvenile loggerhead turtles (Caretta caretta) by analyzing their stable isotope signatures in both the Pacific and Atlantic oceans. Skin samples from oceanic juvenile loggerheads were collected from Peruvian waters in the southeast Pacific and from waters around the Azores Archipelago in the northeast Atlantic and analyzed for delta^15N and delta^13C. Results that turtles in the two ocean regions have delta^13C signatures from -16.3 to 16.7 reflect the oceanic feeding behavior of these loggerhead populations. The delta^15 N signatures in Pacific loggerheads are consistently higher (17.1 0.9) than those of Atlantic loggerheads (7.6 0.5). This inter-ocean difference in delta^15N values was also observed in organisms at the base of the food web in the two study
10 areas. The characteristic predominant process of the nitrogen cycle in each ocean region, which has an effect on the nitrogen composition at the base of the food web, is subsequently transferred to higher trophic levels. Stable isotope signatures in high trophic level organisms, such as oceanic sea turtles, can reveal differences in oceanographic processes.
11 CHAPTER 1 INTRODUCTION Nitrogen fixation and denitrification are the two main processes by which nitrogen flows into and out of the ocean, respectively (Gruber & Sarmiento 1997, Codispoti et al. 2001, Brandes & Devol 2002, Capone & Knapp 2007) (Fig. 1-1). Geographical variations in the natural abundance ratio 14N:15N (expressed as !15N) in marine environments have been explained by distinctive characteristics of the nitrogen cycling in those areas. Low !15N values in food web baseline signatures (Minagawa & Wada 1986, Carpenter et al 1997, Montoya et al. 2002) and nitrogen pools (Wada & Hattori 1976, Li u et al. 1996, Montoya et al. 2002) (Fig. 1-1A) provide evidence of nitrogen fixation, which are observed in the East China, Sargasso, and Arabian Seas, and the western North Atlantic (Minagawa & Wada 1986, Wada & Hattori 1991, Carpenter et al. 1997, Gruber & Sarmiento 1997, Montoya et al. 2002, Deutsch et al. 2007). On the other hand, denitrification evidenced by high !15N values in particulate organic matter (Wada & Hattori 1976, Saino & Hattori 1987, Hobson et al. 1994, Huckstadt et al. 2007), zooplankton (Wu et al. 1997), and inorganic nitrogen (Saino & Hattori 1987, Voss et al. 2001) (Fig. 1-1B) has been observed in oxygen -depleted areas in the eastern tropical North and South Pacific, and the Arabian Sea (Wada & Hattori 1976, Saino & Hattori 1987, Gruber and Sarmiento 1997, Robinson et al. 2007, Deutsch et al. 2007). Even though these differences in !15N at the bas e of the food web in marine systems have been addressed by various studies (references cited above), few have suggested that these baseline differences in !15N are conserved up the food web (Minami & Ogi 1997, Takai et al. 2000, Hatase et al. 2002, Wallace et al. 2006).
12 Stable isotope analysis is an increasingly widespread approach to elucidating the diet, trophic status, foraging habitat and migration patterns in marine animals (Hobson et al. 1994, Rubenstein & Hobson 2004, Revelles et al. 2007, Cherel & Hobson 2007, Newsome et al. 2009). This technique is based on the fact that food webs present a range of stable isotope signatures of nitrogen and carbon that depend mostly, although not exclusively, on the isotopic baseline signature of primary producers supporting that food web. Thus, the tissues of the consumers exhibit an enrichment over their diet of about 3 -5 for !15N and 0 -1 for !13C per trophic level (DeNiro & Epstein 1978, 1981, Minagawa & Wada 1984, Post 2002). However, these commonly used values can be quite variable (Bearhop et al. 2002, Reich et al. 2008) and will depend on the organism and tissues studied (Martnez del Rio et al. 2009). Within a biological community, enrichment in !15N with increasing trophic level makes it useful to infer the trophic status of an animal ( Hobson et al. 1994, Post 2002), while the relatively small enrichment of !13C makes it useful to infer primary sources in a trophic web. In marine ecosystems, !13C is also useful to determine habitat use along three gradients: lowervs. higherlatitude, inshore (neritic) vs. offshore (oceanic), or pelagic vs. benthic (Rau et al. 1982, Hobson et al. 1994, Cherel & Hobson 2007). Endangered loggerhead turtles ( Caretta caretta) are considered generalist predators among sea turtle species (Bjorndal 1997). Recent analysis of diet in oceanic juven ile loggerheads in the central north Pacific and the northeast Atlantic revealed that these turtles are pelagic feeders, foraging opportunistically on pelagic gastropods and barnacles, and cnidarians (Parker et al. 2005, Frick et al. 2009). These studies utilized stomach contents that provided valuable species information of what turtles were
13 feeding on at a particular point of time. Stable isotope analysis provides a complementary tool to address dietary questions with the advantages that it (1) requires a small amount of tissue (e.g., epidermis, blood, epidermal scale) that can be obtained without harming the animal, (2) represents assimilated and not just ingested prey, and (3) quantifies diet over extended periods of time (Hobson et al. 1996), making it a great tool to further understand the trophic ecology of loggerhead turtles. Evaluating the trophic status of a generalist consumer inhabiting known areas of nitrogen fixation or denitrification could provide a broader insight on how these oceanographic processes affect higher trophic levels. When using stable isotopes, it is important to take into account the rate of isotopic incorporation from the diet into the consumer tissue (i.e. discrimination factor), which will depend mainly on the growth and catabolic turnover rate of the consumer tissue (Martnez del Rio et al. 2009). Discrimination factors may be lower and the rate of isotopic incorporation more rapid in fast growing animals than in non-growing animals (Martnez del Rio & Wolf 2005, Reich et al. 2008, Martnez del Rio et al. 2009). Because tissues have different turnover rates, different tissues can reflect dietary information at different temporal scales. High turnover rate tissues such as blood plasma or liver reflect recent diet, while metabolically less active tissue with slower tur nover rates such as skin, bone or scute reflect diet over long-term periods (Rubenstein & Hobson 2004). Turnover rates have been estimated for fast growing, small juvenile loggerhead turtle tissues, with skin tissue showing relatively slow nitrogen and carbon turnover rates of around 45 days, and a !15N discrimination value of 1.60 (Reich et al. 2008).
14 A recent study by Wallace et al. (2006) revealed that leatherback turtles (Dermochelys coriacea) from the Pacific and Atlantic Oceans showed significant differences in the nitrogen isotopic signatures between ocean regions. In the same study, a literature review of !15N signatures revealed a pattern in the nitrogen values between species occupying the same trophic level between denitrification and nitrogen fixation zones. The authors suggested that nitrogen isotopic differences reflected denitrification and nitrogen fixation processes affecting higher trophic levels. Along with the Wallace et al. (2006) study, others have reported similar isotopic results on conspecific species inhabiting different oceanographic areas (Minami & Ogi 1997, Ishibashi et al. 2000, Takai et al. 2000), but none of these conducted food web sampling to corroborate this observation. Building on the study of Wallace et al. (2006), here I evaluate whether the previously described trophic dichotomy in Pacific and Atlantic Ocean regions is evident among other higher trophic organisms and seek to validate the assumption that differences in the baseline !15N signatures are preserved throughout the food web. Because I wanted to eva luate integrated diet in juvenile loggerheads (which would reflect the diet of an opportunistic feeder and therefore their relative trophic status), I used skin tissues that should reflect dietary signatures over an intermediate time frame. I analyzed the stable isotope ratios (!13C and !15N) of juvenile loggerhead turtles, the base of the food web and other food web components in one area of the Pacific and Atlantic oceans with evidence of prevalence of denitrification and nitrogen f ixation processes, respectively. Also, because sampling in the Pacific occurred during a moderate El Nio event, we evaluate if these oceanic juvenile loggerheads can reveal
15 any effect of El Nio event in their isotopic signatures. Finally, I provide insights on the trophic status of juvenile loggerhead turtles as revealed by stable isotope analysis.
16 Figure 1-1 Schematic !15N signatures in the upper water column in the presence of (a) N2 fixation and (b) denitrification. Other processes controlling the isotopic signature are also shown. Solid heavy arrows represent inputs of nitrogen in the water surface through N2 fixation or upwelled nitrates ( NO3 ). Medium solid arrows represent trophic transfer of nitrogen up the web, and light solid arrows represent losses of ingested nitrogen through solid (sinking particles) and dissolved excretes. Uptake of inorganic nitrogen by primary producers is shown by dashed lines (Modified from Montoya et al. 2007).
17 CHAPTER 2 MATERIALS AND METHODS Data and Sample Collection Loggerhead turtle data were collected off southern Peru in the southeast Pacific (SEP) (Fig. 2-1) from December 2003 to May 2007 as part of the small-scale fishery onboard observer program of the Peruvian non-governmental organization Pro Delphinus (Alfaro Shigueto et al. 2008). Turtle data in the northeast Atlantic (NEA) collected from September to October 2002 and August to November 2003 (Fig. 2-2) were obtained during an experimental fishery project evaluating various longline hook designs and the resultant bycatch of loggerhead turtles in the Azores (Bolten et al. 2004). Data reported here are from skin samples of 26 and 53 turtles from the SEP and NEA, respectively. All turtle skin samples were collected from the dorsal surface of the neck of the turtle using a 6 mm biopsy punch or razor blade. Samples were preserved in vials with dry NaCl (SEP) or 70% EtOH (NEA), and neither preservative was found to affect the isotope signatures (Seminoff & Hess, unpub data, Barrow et al. 2008). Data collected for each loggerhead sampled included geographic location (latitude/longitude), date of capture, and curved carapace length (CCL) from the nuchal notch to the posterior-most tip (SEP) or to the posterior marginal notch (NEA) (Bolten et al. 1999). All turtles were released with inconel tags applied to the trailing edge of each fore flipper. In the NEA, food web organisms (e.g., red algae, crabs, barnacles) were gently removed from the carapace of the turtles sampled and preserved in 70% ethanol. Also, zooplankton samples were collected during September 2008 by oblique hauling bongonets (mesh size 200 um) at 50m depth, frozen and then dried at 60C before analysis. In the SEP, tissue samples from other food web organisms (e.g., fish, squid) were
18 collected for analysis during December 2008 with 6mm biopsy punches. To characterize isotopically the base of the food web in the SEP, particulate organic matter (POM, considered the first trophic level in marine environments) samples were also collected off southern Peru in December 2008 by filtration of 4-8 liters of surface seawater through pre-combusted (500 C, 5h) Millipore quartz fiber filters. Food web organisms and POM were stored frozen until dried at 60C prior to sample preparation and analysis. Sample Preparation and Analysis Turtle skin samples were first washed with deionized water and cleaned with alcohol swabs to remove epibionts or any extraneous particles. The outermost layer of the turtle epidermis was separated from the underlying tissue and finely diced with a scalpel blade. Tissue samples from fish and squid were thawed and a small section of muscle was separated from the skin, which was washed in deionized water and then homogenized. For crabs, muscle tissue was obtained from abdominal segments, and for barnacles, whole organisms (not including the shells) were analyzed. Barnacle tissues were soaked in 1N HCl to remove carbonates. Red algae samples were rinsed with deionized water and small invertebrates were removed. All turtle skin, fish, squid, crab, barnacle, algae and zooplankton samples were dried at 60C for 24 hours. Lipids were extracted from turtle, fish, squid, crab, and zooplankton samples using petroleum ether in a Dionex accelerated solvent extractor. All samples, except turtle samples, were then ground to a fine powder using a mortar and pestle. POM filter samples were dried at 60C for 24 hours and washed with 1N HCl to remove carbonates and dried again for analysis.
19 For stable isotope analysis, approximate ly 500-600 g of each sample was weighed and sealed in a tin capsule. Between 1200-1500 g was loaded for red algae. POM filters were cut into halves that were also loaded in tin capsules. Samples were then analyzed for carbon and nitrogen stable isotope ratios by combustion in a COSTECH ECS 4010 elemental analyzer interfaced via a Finnigan-MAT ConFlow III device (Finnigan MAT, Breman, Germany) to a Finnigan-MAT DeltaPlus XL (Breman, Germany) isotope ratio mass spectrometer in the Stable Isotope Geochemistry Lab at the University of Florida, Gainesville, FL, USA. Stable isotope ratios, that compare the ratios of heavy and light isotopes of an element in the sample to an international standard, are reported in the conventional !notation: !X = [(Rsample/Rstandard) -1] x 1000 where !X is the relative abundance of 13C or 15N in the sample expressed in parts per thousand (); Rsample and Rstandard are the ratios of heavy to light isotope (13C/12C and 15N/14N) in the sample and international standard, respectively. The standard used for 13C was Vienna Pee Dee Belemnite and for 15N was atmospheric N. Working standards L-glutamic acid USGS40 (!13C = -26.23 and !15N = -4.52) were calibrated monthly against international standards and were inserted in all runs at regular intervals to calibrate the system. The analytical accuracy of our measurements, measured as the SD of replicates of standards, was 0.20 for !13C and 0.14 for !15N (n = 28 and 28, respectively).
20 Statistical Analysis Sea turtle data were tested for normality using the Shapiro-Wilk test. Carbon and nitrogen stable isotope signatures in turtle skin samples were compared between ocean regions and among years within ocean regions with Students t-tests. For evaluation of nitrogen and carbon stable isotope signatures among years within ocean regions, I compared values for turtles sampled in the Pacific in austral summer (December to March) 2003-2004 and 2006-2007. Atlantic samples were compared between samples collected during September-October 2002 and 2003. The relationships between stable isotope values and body size were evaluated with linear regressions. The software program R (http://www.r-project.org/) was used to analyze stable isotope and sea turtle data. An le vel of 0.05 was used to evaluate the significance of all tests.
21 Figure 2-1. Collection sites of turtle skin (open circles), particulate organic matter (filled squares), and other organisms (crosses) samples off southern Peru in the southeast Pacific.
22 Figure 2-2. Collection sites of turtle skin (open triangles), red algae (filled diamonds), and other organisms (crosses) samples in Azorean waters in the northeast Atlantic.
23 CHAPTER 3 RESULTS Values of !13C from SEP loggerhead samples ranged from -14.6 to -17.4 and NEA loggerhead samples ranged from -15.7 and -17.8 ; while values for !15N ranged from 15.2 to 18.7 in SEP samples and from 6.4 to 8.7 in NEA samples. The !13C and !15N values for loggerheads were normally distributed (Shapiro-Wilk test, p > 0.05). Although the NEA !13C values were almost completely encompassed by the SEP values (86% overlap of the data), NEA !13C values were significantly lower than those from the SEP (mean SD, SEP = -16.3 0.7 NEA = -16.7 0.5 t = 3.7, p < 0.001, Fig. 3-1). Also, SEP samples showed significantly higher !15N values than those from the NEA (mean SD, SEP = 17.1 0.9, NEA = 7.6 0.5, t = 59.3, p < 0.001, Fig. 3-1). In the SEP, POM samples had the lowest !15N (12.1 1.2) and !13C (-22.4 1.7) values (Table 3-1, Fig. 3 -2). Other oceanic organisms had !15N values between 14.9 1.6 (Mahi mahi Coryphaena hippurus) and 18.6 0.4 (jumbo squid Dosidicus gigas) and !13C values from -16.4 (juvenile blue shark Prionace glauca) to 18.0 0.3 (flying fish Exocoetus sp.) (Table 3-1, Fig. 3 -2). A similar scenario was found in the NEA with red algae presenting the lowest !15N (3.7 0.7) and !13C (-20.6 0.7) values. Other food web components ranged from 4.4 0.6 to 9.0 0.6 for !15N and -20.4 0.3 and -18.41 0.6 for !13C, with lowest values found in zooplankton and highest values found in Columbus crab Planes minutus (Table 3-1, Fig. 3 -2).
24 The difference in mean !15N values between primary producers (POM and red algae) and loggerheads was 4.9 and 4.0, while !13C varied by 6.2 and 3.9, in the SEP and NEA, respectively. It is important to note that red algae do not reflect the base of the food web in the open ocean (unlike POM) and is only used as a proxy for baseline isotopic signature. However, I might expect POM isotopic nitrogen and carbon values in the NEA to be lower than those of red algae (as I will explain below). Additionally, POM values are used here as approximates of what the baseline signatures in the SEP are, since they were sampled in a different year from those of SEP turtle samples. The difference of ~0.9 (4.9 4.0) between variations in !15N for primary producers and loggerheads is lower than the 15N discrimination value per trophic level for juvenile loggerheads (1.60, Reich et al. 2008); thus, I estimate that the relative trophic position in both loggerhead populations should be similar. Turtles sampled in the SEP ranged from 35.9 to 81.4 cm curved carapace length (CCL). Size range for loggerheads in the area is consistent with the size range previously reported by Alfaro Shigueto et al. (2008). This size distribution corresponds to i mmature individuals according to criteria cited by Limpus & Limpus (2003). I found a significant positive correlation between !15N and body size (R2 = 0.26, df = 24, p = 0.008, Fig. 3-3). However, no statistically significant relationship was found for !13C and turtle size (p = 0.354). Turtles in the NEA ranged in size from 33.1 to 80.3 cm CCL. According to the minimum adult size criteria by Bjorndal et al. (2000), all of our turtles correspond to juvenile individuals. Similar to loggerheads in the SEP, a significant positive (although weak) relationship was found between !15N and body size (R2 = 0.10,
25 df = 51, p = 0.023, Fig. 3-3), and the relationship found between !13C and body size was not significant (p = 0.110). Significant annual variation was found in loggerheads for !13C in the SEP and for both !13C and !15N in the NEA. In the SEP, !13C values for samples collected during 2006-07 were significantly depleted relative to samples gathered in 2003-04 (2003-04 = -15.8 0.7, 2006 -07 = -16.5 0.4, t = 2.9, p = 0.004), but data showed a 67% overlap. In the NEA, 2002 had significantly higher !15N values than 2003 (mean SD, 2002 = 8.0 0.4, 2003=7.5 0.5, t = 3.6, p < 0.001) but with 84% overlap of data. Carbon (!13C) values were more depleted in 2003 (mean SD, 2002 = -16.2 0.3, 2003 = -17.0 0.4, t = 7.2, p < 0.001).
26 Table 3 -1. Stable isotope ratios (!15N and !13C) of marine organisms in the southeast Pacific and the northeast Atlantic. Values are mean SD n 15 N( ) 13 C( ) Source SOUTHEAST PACIFIC POM N/A 12.1 1.2 22.4 1.7 this study Mollusca Jumbo squid 3 18.6 0.4 17.6 0.4 this study Chondrychthyes Blue shark 1 18.2 16.4 this study Osteichthyes Anchovy 2 14.2 0.9 16.7 0.1 Catenazzi & Donelly 2008 Mahi mahi 2 14.9 1.6 16.5 0.1 this study Flying fish 3 16.9 0.9 18.0 0.2 this study Mackerel 2 15.8 1.4 17.0 0.8 this study Reptilia Loggerhead turtle 26 17.1 0.9 16.3 0.7 this study Leatherback turtle 17 15.4 1.8 19.1 0.7 Wallace et al. 2006 Mammalia Sea lion 6 17.4 0.9 15.1 0.4 Catenazzi & Donelly 2008 Sperm whale 12 18.6 to 20.3 16.0 to 14.7 Marcoux et al. 2007 NORTHEAST ATLANTIC Red algae 5 3.7 0.7 20.6 0.7 this study Zooplankton 3 4.4 0.6 20.4 0.2 this study Crustacea Acorn barnacle 3 7.6 0.1 19.0 0.6 this study Goo seneck barnacle 4 5.7 1.6 20.0 0.5 this study Pedunculate barnacle 3 5.7 0.7 19.1 0.6 this study Columbus crab 5 9.0 0.6 18.4 0.5 this study Reptilia Loggerhead turtle 53 7.6 0.5 16.7 0.5 this study Leatherback tu rtle 9.8 1.5* 10.3** 10.4 + 19.4 1.0* 19.6 ** 19.5 + Wallace et al. 2006 Maros et al. 2006 Caut et al. 2008 Aves Corys shearwater 35 13.2 16.2 Ramos et al. 2009 Mam malia Sperm whale 1 13.2 11.8 Mendes et al. 2007 Leatherbac k turtle values are from nesting females in *St Croix, Virgin Islands and **+French Guiana in the Atlantic
27 Figure 3-1. Stable isotope ratios of nitrogen ( !15N) and carbon (!13C) of skin samples from juvenile loggerheads Caretta caretta off southern Peru in the southeast Pacific (open circles) and in Azorean waters in the northeast Atlantic (open triangles). POM values off southern Peru, and red algae and zooplankton samples from Azorean waters are also shown.
28 Figure 3-2. Comparison of stable isotopes ratios of the food web components in the southeast Pacific (SEP; open circles) and in the northeast Atlantic (NEA; solid triangles). All SEP values are from waters off Peru except for those of leatherback sea turtles. Leatherback isotope values are from Wallace et al. (2006), those of anchovy and sea lion are from Catenazzi & Donnelly (2008), and those of sperm whale are from Marcoux et al. (2007). In the NEA, leatherback turtle isotopic values are from post-nesting females in St Croix, Virgin Islands in the Atlantic (Wallace et al. 2006). Values of sperm whale are from Mendes et al. (2007) and those from Corys shearwater are from Ramos et al. (2009). All values are mean range. 1. POM (SEP) or Red algae (NEA), 2. Zooplankton, 3. Gooseneck barnacle Lepas anatifera, 4. Pedunculate barnacle Conchoderma virgatum 5. Acorn barnacle, 6. Loggerhead turtle Caretta Caretta, 7. Columbus crab Planes minutus, 8. Leatherback turtle Dermochelys coriacea, 9. Corys shearwater Calonectris diomedea borealis 10. Sperm whale Physeter macrocephalus 11. Anchovy Engraulis ringens, 12. Mahi mahi Coryphaena hippurus., 13. Chub mackerel Scomber japonicus, 14. Flying fish Exocoetus sp., 15. Sea lion Otaria flavescens, 16. Blue shark Prionace glauca, 17. Jumbo squid Dosidicus gigas.
29 Figure 3-3. Skin !15N vs. curved carapace length in loggerhead sea turtles Caretta caretta off (a) southern Peru in the southeast Pacific and (b) the Azores in the northeast Atlantic. Both relationships are significant, see text.
30 CHAPTER 4 DISCUSSION Differences between Ocean Regions Based on satellite telemetry studies, juvenile loggerheads in the NEA feed in oceanic, epipelagic zones (Santos et al. 2008). SEP juvenile loggerheads analyzed in this study were all captured in the oceanic zone (Alfaro Shigueto et al. 2008). I would expect to find similar !13C in both SEP and NEA loggerhead populations reflecting their oceanic feeding behavior. Although !13C values were significantly depleted for NEA loggerheads, carbon value ranges for both populations greatly overlap (86%) confirming their oceanic and pelagic feeding strategy (Fig. 3-1). Overall, lower !13C in NEA loggerheads may be explained by the latitudinal variation observed in marine phytoplankton !13C, which decreases from the equatorial zones toward the polar regions (Rau et al. 1982, Goericke & Fry 1994). This baseline latitudinal variation in !13C has been reflected in higher consumers such as squids and penguins (Takai et al. 2000, Cherel & Hobson 2007). However, this decline is more pronounced in the southern versus northern hemisphere (-0.14 and -0.015 per degree in the southern and northern hemisphere, respectively) and other factors may be playing a role for the significant variation in carbon signatures. In our study, red algae !13C values in the Azores (38N) were more enriched than values from POM off southern Peru (17S). Red algae in the NEA do not necessarily reflect the main primary producer in the open ocean (as phytoplankton does) but serve as a proxy for a baseline isotopic signature in Azorean waters. Variations in the thickness of the diffusive boundary layer between phytoplankton (POM) and macroalgae (red algae) affect the diffusion of carbon during carbon uptake (France 1995). This may translate into more depleted !13C signatures
31 for phytoplankton compared to macroalgae because heavy isotopes (13C) are easily discriminated through the thin boundary layer of the former. Thus, expected !13C values of POM from the Azores should be depleted relative to red algae which may explain the more depleted !13C values in NEA loggerheads. Our study shows that !15N values in SEP loggerheads were 9.4 higher than NEA loggerhead values (Fig. 3-1). While !15N is useful to determine the relative trophic position of organisms within a community (Post 2002, Newsome et al. 2007), species with known relative trophic status allow for comparison of their nitrogen signatures between ecosystems. Large differences in the nitrogen signature of organisms suggests differences in trophic levels, but diet studies in oceanic juvenile loggerhead populations have shown that they are opportunistic predators, and as a result, both populations should occupy a similar trophic level. For example, in Azorean waters and the central north Pacific oceanic juvenile loggerhead turtles feed on various pelagic organisms such as jellyfish, sea slugs, sea snails and pelagic barnacles (Parker et al. 2005, Frick et al. 2009), which are prey that generally feed at lower trophic levels. Additionally, similar !15N variation between primary producers and loggerhead populations reported in this study suggest that they occupy a similar relative trophic level. Therefore, I attribute this significant !15N difference between SEP and NEA loggerhead populations to different !15N values at the base of the food chain. Nitrogen isotopic signatures at the base of the food web are affected by characteristic nitrogen cycle dynamics in the marine environment (Saino & Hattori 1987, Carpenter et al. 1997, Montoya et al. 2002). In the tropical North Atlantic, nitrogen fixation is the main process by which nitrogen is assimilated (Carpenter et al. 1997,
32 Montoya et al. 2002). However, few studies have addressed the characteristics of the nitrogen dynamics in the northeast Atlantic, specifically in the waters of the Azores region. Bourbonnais et al. (2009) analyzed the !15N distribution in the water column in the Azores Front region. They found clear evidence for nitrogen fixation and reported dissolved organic nitrogen !15N values of approximately 2.6. Because the isotope fractionation during nitrogen fixation is small and atmospheric N is near 0, slightly depleted values of !15N have been reported for organic matter (Fig. 1a). Thus, expected !15N values of POM in the Azores should be close to 0 if nitrogen were mainly supplied by nitrogen fixation (Minagawa & Wada 1986, Carpenter et al 1997, Montoya et al. 2002) or relatively low if other sources of nitrogen were available (e.g., upwelled nitrates, Montoya et al. 2002). The !15N of red algae was 3.7, which may reflect the uptake of other sources of nitrogen besides that from nitrogen fixation. However, as mentioned above, red algae were used as a proxy for isotopic baseline signature in the Azores; expected POM !15N may be lower than that from the macroalgae. Differences in nitrogen uptake rates due to physiological characteristics of phytoplankton and macroalgae (Montoya & McCarthy 1995, Needoba et al. 2003, De Brabandere et al. 2007) may render significantly divergent !15N signatures (Hobson & Welch 1992, Bode et al. 2006). Phytoplankton discriminates against the heavy nitrogen isotope (15N) during nitrate uptake, with an average isotopic discrimination value of ~ 5 (Montoya & McCarthy 1995, Needoba et al 2003) while discrimination values for macrophytes are suggested to be lower (DeBrabandere et al. 2007). This translates to more depleted !15N values in phytoplankton than in macroalgae. Moreover, the !15N of zooplankton, one trophic step above POM, in our study was 4.4 (Fig. 3-1) which is
33 similar to values reported for zooplankton in the nitrogen fixation tropical North Atlantic (Montoya et al. 2002). The SEP is characterized by a shallow oxygen-depleted water layer (~50-500 m) off Peru, where high rates of denitrification occur (Tarazona et al. 2003, Robinson et al. 2007, including autotrophic denitrification: Ward et al. 2009). As nitrate is consumed during denitrification, lighter isotopes of nitrogen are preferentially utilized, leaving residual nitrate pools heavily enriched in 15N. The resultant high !15N in the subsurface denitrification layer is transmitted to surface waters through upwelling (Saino & Hattori 1987, Wada & Hattori 1991, Robinson et al. 2007). The constant supply of 15N enriched nitrates in the surface ocean are later assimilated by phytoplankton (Fig. 1b) which in turn show high !15N signatures, as reflected in our POM samples (Fig. 3-1). Deutsch et al. (2007) recently evaluated the rates of nitrogen fixation in the marine environment through a novel analysis of ocean surface nutrients. They found that the highest rates of nitrogen fixation are geographically coupled with that of denitrification. In other words, the highest rates of nitrogen fixation are found in the Eastern Tropical Pacific (ETP where highest rates of denitrification occur) and not in the tropical North Atlantic as previously thought. However, net rates of denitrification in the ETP are much higher than fixation so that they obscure the isotopic evidence of nitrogen fixation, as reflected in the high values of !15N in our POM samples. It is not certain if, at a much finer scale, areas of nitrogen fixation in the ETP overlap with that of denitrification. It is assumed that the former areas are close to these high denitrification, high nutrient waters, but whether there could be an overlap or not has not yet been revealed (Deutsch et al. 2007).
34 The !15N dichotomy revealed in loggerhead populations can be therefore explained by the variations in nitrogen signatures at the base of the food web. To further assess the effect of highest rates of nitrogen fixation and denitrification on the food web, I compiled !15N and !13C values in primary producers and other organisms of the food web in the SEP and in the NEA (Fig. 3-2). Isotopic signatures in the food web of the SEP show that high nitrogen baseline values driven by denitrification are conserved through several trophic levels. A similar pattern is observed in the NEA, where low !15N in red algae and especially zooplankton due to nitrogen fixation give the food web overall low !15N values when compared to the isotopic values in the SEP food web. Trophic structure analysis was not performed due to the limited number of individuals and/or species sampled, different sampling dates, and/or the lack of predator prey related signatures (e.g., those of loggerheads and potential prey). However, organisms analyzed in this study, as well as the collection of literature isotopic signatures of organisms off southern Peru and the Azores, allows for a first insight in to the isotopic composition of the food web structure off the SEP and the NEA. In the SEP, average !15N signature of anchovy Engraulis ringens is low which reflects its diet preference in the area of large zooplankton (Espinoza & Bertrand 2008). The !15N increases in larger fishes such as mackerel Scomber japonicus and flying fish Exocoetus spp. Also as expected, higher trophic level species such as sea lion Otaria flavescens, sperm whale Physeter macrocephalus, blue shark Prionace glauca and jumbo squid Dosidicus gigas show much higher !15N signatures (Fig. 3-2). A similar pattern is observed in the Azores, where nitrogen signatures in filter feeding organisms
35 such as pedunculate barnacles ( Conchoderma virgatum and Lepas anatifera) show higher values than those from zooplankton but lower than those from crabs or loggerhead turtles. Highest !15N value s are presented by sperm whale Physeter macrocephalus and Corys shearwater Calonectris diomedea borealis which reflects their diet preference of mainly cephalopods and fish, respectively (Clarke et al. 1993, Granadeiro et al. 1998). The difference in !15N signatures between Pacific and Atlantic loggerheads found in this study was higher than that reported by Wallace et al. (2006) between leatherbacks in the same ocean regions (9.5 and 5.6 for loggerheads and leatherbacks, respectively, Fig. 3-2). In fact, when comparing !15N values between species, loggerhead !15N values are higher than leatherback !15N values in the Pacific (mean SD, loggerheads = 17.1 0.9; leatherbacks = 15.4 1.8), but lower in the Atlantic populations (mean SD, loggerheads = 7.6 0.5; leath erbacks = 9.8 1.5). The specialist feeding behavior of leatherbacks on jellyfish and the generalist feeding strategy of loggerheads, with commonly known intermediate and higher trophic status respectively, allows the comparison of nitrogen signatures between these two species. The higher !15N in Atlantic leatherbacks than in Atlantic loggerheads (also reported by Maros et al. 2006 and Caut et al. 2008) may be reflecting different oceanic regions utilized by these species. I suggest that nesting leatherbacks from St. Croix may be foraging in areas with low or no nitrogen fixation activity, where baseline signatures !15N are not close to zero. On the other hand, the !15N values for loggerheads and leatherbacks in the Pacific would be reflecting the feeding strategies of these turtles revealed by conventional studies (Bjorndal 1997, Parker et al. 2005, Frick
36 et al. 2009) considering they were foraging in the same feeding ground or at least in waters with similar baseline 15N. Most post-nesting leatherback turtles from the Pacific coast of Costa Rica migrate to oceanic foraging areas in the South Pacific gyre characterized by low productivity (Shillinger et al. 2008). However, early reports on eastern Pacific leatherback migration and current data on bycatch in fisheries off Peru reveal that these leatherbacks also utilize high-productivity waters off Peru (Eckert & Sarti 1997, Alfaro-Shigueto et al. 2007). Data compiled by Suryan et al. (2009) across a wide range of trophic levels in several oceanic regions suggest that environmental forces on resource availability drive variations in the life history of conspecifics. To what extent the differences in energy flow in our study areas (through prevalence of nitrogen fixation in one and denitrification in the other), along with other climatic factors affecting nutrient availability, may shape the biology of these juvenile loggerhead populations is still poorly understood. Interannual Variation of Loggerhead !13C and !15N within Ocean Regions The geographic variability observed in !15N of primary producers and POM due to distinct marine nitrogen cycles may be affected by oceanographic/atmospheric changes (Wada & Hattori 1991). In particular, the SEP experiences El Nio-Southern Oscillation (ENSO) cycles with its warm El Nio (EN) phase. The main biological consequences of EN events in the SEP (particularly off Peru) are linked to the decline of primary productivity which produces a chain reaction that ultimately affects survival, reproduction, and distribution of species at higher trophic levels (reviewed in Wang & Fiedler 2006). Some of our samples from the SEP were collected during a moderate EN event (season 2006-2007; n = 13) which allowed for comparison with a normal
37 condition season (2003-2004; n = 9). There is significant variability between seasons 2003-04 and 2006 -07, as evidenced by the slightly more depleted values for !13C in 2006-07. However, no significant effect of EN event was found on the !15N values in loggerheads as expected. Because the thermocline and oxygen-depleted layer deepens and the upwellings cease during EN events, which disrupt the transport of nutrient -rich waters (including 15N enriched nitrate) to the surface, primary production declines significantly (Tarazona et al. 2003). Hence, I had expected to find significantly reduced !15N values in the 2006-07 loggerhead turtles. The EN event of 2006-07 may not have had a strong effect on the !15N, as a result of its moderate magnitude. In fact, after the strong 1997-1998 EN event, favorable conditions for upwelling of highly productive waters have not been affected by recent moderate EN events (Marzloff et al. 2009). On the other hand, due to the appearance of warmer water masses, EN events in the SEP are also characterized by a shift in phytoplankton composition from mainly centric diatoms to dinoflagellates (Sanchez et al. 2000, Tarazona et al. 2003). This shift could entail a !13C variation in the primary producers with further effect up the food web, and it could explain the depleted !13C values observed in the 2006-07 loggerheads. A !13C depletion in zooplankton has been observed in waters in which fast-growing centric diato m production was low and presence of slow-growing dinoflagellates was high (Fry & Wainwright 1991). Popp et al. (1998) suggested that phytoplankton cell geometry, as well as growth rate, had an effect on their !13C signature. Phytoplankton with higher surface-area-to-volume ratios (e.g., pennate diatoms, dinoflagellates) have higher isotopic fractionation values associated with photosynthesis and thus more depleted
38 !13C, because photosynthesis discriminates against 13C. Further investigation of variation of isotopic signatures should be conducted with stronger ENSO effects. I found interannual variation in the !15N and !13C of loggerheads in the NEA, although the differences were relatively small in both cases. The waters off the Azores are influenced by the large-scale North Atlantic Oscillation (NAO) with reported effects on abundance, biomass, and distribution of marine fauna due to variation in availability of food resources (Drinkwater et al. 2003). The interannual variation observed in the isotopic signatures in loggerheads, however, cannot be explored in relation to NAO, as this is a long-term event for which one would need a larger data set to analyze. When accounting for other factors that may explain this significant interannual variation in th e isotopic signatures such as turtle geographic position or sea surface temperature, I did not find any correlation (data not presented). Variability of !15N in organisms can be a result of changes in their trophic positions or changes in the baseline !15N signatures. I lack baseline signatures in the years when turtles in the NEA were sampled. However, assuming baseline nitrogen signatures did not differ between years, I may suggest that 2002 loggerheads presented a relative higher trophic level than 2003 loggerheads. Marked seasonality in primary production with subsequent effects on isotopic signatures is expected in temperate and arctic systems than in tropical and subtropical systems (Tamelander et al. 2009). Nevertheless, Azorean waters are characterized by a high activity of eddies (Le Traon & De Mey 1994). These oceanographic phenomena have known effects on higher trophic levels through enhancement of primary production and biomass (Falkowski et al. 1991, Oschlies & Garon 1998). Satellite transmitter studies conducted by M. Santos
39 (unpublished data) suggest that the foraging behavior of loggerheads in Azorean waters is related to eddies. Indeed, because of the opportunistic feeding behavior by loggerheads (Frick et al. 2009), enhancement of productivity due to eddies may allow them to consume a great variety of organisms, including several of higher trophic levels (which will translate in higher loggerhead !15N). Additionally, eddies may also change the phytoplankton composition (Lochte & Pfannkuche 1987, Vaillancourt et al. 2003) which in return will have an effect on !13C signature (Fry & Wainwright, 1991). However, although the nature and frequency of eddies in Azorean waters have been investigated (Mourio et al. 2003), their effect on the marine fauna has rarely been studied (Huskin et al. 2001) and thus, I can only speculate on their effect for loggerhead foraging ecology and isotopic signatures reported here. Further compound-specific nitrogen isotope analysis of loggerhead tissues could help elucidate whether the differences in !15N are due to metabolic and trophic-level relationships or changes in isotopic composition at the base of the food web without the necessity of food web base sampling (Popp et al. 2007). Relationship between !15N and Turtle Size Variability of !15N in or ganisms within an ecosystem generally suggests changes in trophic levels. However, it may also reflect differences in the nitrogen isotopic composition at the base of the food web due to temporal variation of the !15N of nutrients. These need to be long term variations to have an effect on the isotopic composition of higher trophic level or larger animals with low turnover rates for nitrogen (Montoya et al. 2007 ). Additionally, within ecosystem spatial variation of the isotopic composition of nutrients has been observed in deeper waters (Saino & Hattori 1987,
40 Montoya et al. 2002). Microbial and zooplankton consumption generate 15N-enriched residual matter (Saino & Hattori 1987) that becomes the primary food source for deepwater organisms. Thus, mesopelagic organisms may have overall higher !15N values (Rau et al. 1989), if one assumes that baseline signatures throughout the water column are conserved up higher trophic levels (Graham et al. 2007). I lack baseline !15N signatures in the years when turtle samples were collected and/or information on prey items consumed by these turtles to better determine whether the !15N differences observed are due to trophic change or temporal variation of baseline signatures. However, the increase of loggerhead !15N with size may be based on three facts: turtle (1) growth rate, (2) gape size, and/or (3) feeding on mesopelagic prey. Rapidly growing smaller juveniles (with higher growing rates) may show a lower nitrogen discrimination value than larger juveniles (Martnez del Rio et al. 2005, Reich et al. 200 8). This translates to lower !15N values in smaller juveniles even if they are feeding on the same diet as larger juveniles. Nevertheless, larger turtles could be feeding on higher trophic level organisms. Factors such as gape size may be playing an important role in determining access to greater diversity and/or size of prey items accessible for larger turtles. Allometry in the head of loggerhead turtles reported by Kamezaki & Matsui (1997) may allow turtles to feed on bigger pelagic items of higher trophic level. A higher trophic level in larger turtles has been already observed in juvenile loggerheads in the Atlantic that move from oceanic to developmental neritic zones where they feed mainly on demersal prey items (Bolten 2003). However, juvenile loggerheads in the SEP and NEA inhabit exclusively oceanic waters (>200 m depth, Alfaro Shigueto et al. 2008, Santos et al. 2008) where benthic feeding is not likely to
41 occur. Additionally, stomach studies in oceanic juveniles do not seem to support a higher trophic level for larger turtles. Even though Frick et al. (2009) found a greater diversity of prey species in the stomachs of larger loggerheads in Azorean waters, they argued that this could be due to the larger volume of stomach contents in larger turtles rather than a more diverse diet. Moreover, Parker et al. (2005) found that there was no relation between the volume of stomach content or size of the prey ingested and the size of the turtles (35-70 cm CCL) in the central north Pacific. The third explanation for the enriched loggerhead !15N in larger turtles may be related to the consumption of mesopelagic organisms with enriched !15N values. Because POM !15N increases with depth (Saino & Hattori 1987, Montoya et al. 2002), feeding on mesopelagic prey would increase the !15N in turtles compared to those with an epipelagic feeding behavior. Mesopelagic prey items have been found in stomach contents in oceanic loggerheads in the central north Pacific (Parker et al. 2005). This feeding behavior has been also observed through satellite transmitter studies by showing that oceanic loggerheads can dive and actively forage up to 100 m depth (Polovina et al. 2003). In any case, stable isotopes generally cannot pinpoint the specific diet items consumed and therefore, knowledge and sampling of potential prey items is required to accurately explain trophic changes in loggerheads.
42 CHAPTER 5 CONCLUSIONS In this study, loggerhead turtles revealed the ocean region differences in !15N that were also found in leatherback turtles, while !13C were similar between turtle populations, confirming their oceanic foraging behavior. The dichotomy in the !15N at the base of the food web and other organisms within the SEP and NEA informs us that this difference in the baseline nitrogen signature is conserved through higher trophic levels, thus reflecting characteristics of the nitrogen cycling regime in each ocean region. Stable isotopes of carbon and nitrogen in higher trophic level organisms, such as sea turtles, present the potential to elucidate changing oceanographic conditions, but systematic and long-term sampling is needed. Also, the fact that both denitrification and nitrogen fixation occur at highest rates in the ETP lead us to formulate some questions: is this pattern observed in loggerheads and the food web of the SEP due to denitrification alone? To what extent does denitrification and not nitrogen fixation affect !15N signatures? Can changing oceanographic conditions such as EN events with effects on the extent of denitrification rates reveal differences in the nitrogen cycle and can this be reflected in higher organisms? Additional compound-specific isotopic analyses may explain variations in nitrogen source and hence help reveal changing oceanographic conditions.
43 LIST OF REFERENCES Alfaro Shigueto J, Mangel JC, Seminoff JA, Dutton PH (2008) Demography of loggerhead turtles Caretta caretta in the southeastern Pacific Ocean: fisheries-based observations and implications for management. Endan Species Res 5:129 -135 Alfaro-Shigueto J, Dutton PH, Van Bressem MF, Mangel J (2007) Interactions between leatherback turtles and Peruvian artisanal fisheries. Chelonian Conserv Biol 6:129 134 Barrow L, Bjorndal K, Reich K (2008) Effects of preservation method on stable carbon and nitrogen isotope values. Physiol Biochem Zool 81:688-693 Bearhop S, Waldron S, Votier SC, Furness RW (2002) Factors that influence assimilation rates and fractionation of nitrogen and carbon stable isotopes in avian blood and feat hers. Physiol Biochem Zool 75:451 -458 Bjorndal KA (1997) Foraging Ecology and Nutrition of Sea Turtles. In: Lutz PL, Musick JA (eds) The biology of sea turtles. Boca Raton, Fla: CRC Press, p 199-222 Bjorndal K, Bolten A, Martins H (2000) Somatic growth model of juvenile loggerhead sea turtles Caretta caretta: duration of pelagic stage. Mar Ecol Prog Ser 202:265-272 Bode A, Alvarez-Osorio MT, Varela M (2006) Phytoplankton and macrophyte contributions to littoral food webs in the Galician upwelling estimated from stable isotopes. Mar Ecol Prog Ser 318:89102 Bolten AB (1999) Techniques for measuring sea turtles. In: Eckert KL, Bjorndal KA, Abreu-Grobois FA, Donelly M (eds) Research and Management Techniques for the Conservation of Sea Turtles. IUCN/SSC Marine Turtle Specialist Group Publication No. 4, p 126 -132 Bolten AB (2003) Active swimmers-passive drifters: the oceanic juvenile stage of loggerheads in the Atlantic system. In: Bolten AB, Witherington BE (eds) Loggerhead sea turtles. Washington, DC. Smithsonian Institution Press, p 63-78 Bolten AB, Martins HR, Isidro E, Santos M, Ferreira M, Bettencourt E, Giga A, Cruz A, Bjorndal KA (2004) Experiment to evaluate gear modification on rates of sea turtle bycatch in the swordfish longline fishery in the Azores Phase 1 and Phase 2. In Long KJ and Schroeder BA(eds), Proceedings of the International Technical Expert Workshop on Marine Turtle Bycatch in Longline Fisheries, Seattle, Washington, 1113 February 2003. NOAA Technical Memorandum NMFS-OPR-2, p 239 -253 Bourbonnais A, Lehmann MF, Waniek JJ, Schulz-Bull DE(2009) Nitrate isotope anomalies reflect N2 fixation in the Azores Front region (subtropical NE Atlantic) J Geo Res 114: C03003, doi:10.1029/2007JC004617
44 Brandes JA, Devol AH (2002) A global marine-fixed nitrogen isotopic budget: Implications for Holocene nitrogen cycling Global Biogeochem Cycles 16:1120, doi:10.1029/2001GB001856 Capone G, Knapp A (2007) A marine nitrogen fix? Nature 445:159-160 Carpenter EJ, Harvey HR, Fry B, Capone DG (1997) Biogeochemical tracers of the marine cyanobacterium Trichodesmium. Deep-Sea Res I 44:2738 Catenazzi A, Donnelly M (2008) Sea lion Otaria flavescens as host of the common vampire bat Desmodus rotundus. Mar Ecol Prog Ser 360:285-289 Caut S, Guirlet E, Angulo E, Das K, Girondot M (2008) Isotope analysis reveals foraging area dichotomy for Atlantic leatherback turtles. PLoS ONE 3(3):e1845. doi:10.1371/journal.pone.0001845 Cherel Y, Hobson KA (2007) Geographical variation in carbon stable isotope signatures of marine predators: a tool to investigate their foraging areas in the Southern Ocean. Mar Ecol Prog Ser 329:281-287 Clarke MR, Martins HR, Pascoe P (1993) The diet of sperm whales (Physeter macrocephalus Linnaeus 1758) off the Azores. Phil Trans R Soc Lond B 339:67-82 Codispoti LA, Brandes JA, Christensen JP, Devol AH, Naqvi SWA, Paerl HW, Yoshinari T (2001) The oceanic fixed nitrogen and nitrous oxide budgets: Moving targets as we enter the anthropocene? Sci Mar 65:85 -105 De Brabandere L, Frazer TK, Montoya J (2007) Stable nitrogen isotope ratios of macrophytes and associated periphyton along a nitrate gradient in two subtropical, spring-fed streams. Fresh Biol 52:156-1575 DeNiro MJ, Epstein S (1978) Influence of diet on the distribution of carbon isotopes in animals. Geochim Cosmochim Acta 42:495506 DeNiro MJ, Epstein S (1981) Influence of diet on the distribution of nitrogen isotopes in animals. Geochim Cosmochim Acta 45:34135 Deutsch C, Sarmiento JL, Sigman DM, Gruber N, Dunner JP (2007) Spatial coupling of nitrogen inputs and losses in the ocean. Nature 445:163-167 Drinkwater KF, Belgrano A, Borja A, Conversi A, Edwards M, Greene CH, Ottersen G, Pershing AJ, Walker H (2003) The response of marine ecosystems to climate variability associated with the North Atlantic Oscillation. In: Hurrell JM, Kushnir Y, Ottersen G, Visbeck M (eds) The North Atlantic Oscillation: Climatic Significance and Environmental Impact, American Geophysical Union p 211234
45 Eckert SA, Sarti L (1997) Distant fisheries implicated in the loss of the worlds largest leatherback nesting population. Mar Turt Newslet 78:27 Espinoza P, Bertrand A (2008) Revisiting Peruvian anchovy ( Engraulis ringens) trophodynamics provides a new vision of the Humboldt Current system, Prog Oceanogr 79:215227 Falkowski PG, Ziemann D, Kolber Z, Bienfang P (1991) Role of eddy pumping in enhancing primary production in the ocean. Nature 352:5558 France RL (1995) Carbon-13 enrichment in benthic compared to planktonic algae: foodweb implications. Mar Ecol Prog Ser 124:307-312 Frick MG, Williams KL, Bolten AB, Bjorndal KA, Martins HR (2009) Foraging ecology of oceanic-stage loggerhead turtles ( Caretta Caretta ), opportunistic carnivores. Endan Species Res 9:9197 Fry B, Wainwright S (1991) Diatom Sources of C-13-rich carbon in marine food webs. Mar Ecol Prog Ser 76:149-157 Goericke R, Fry B (1994) Variations of marine plankton !13C with latitude, temperature, and dissolved CO2 in the world ocean. Global Biogeochem Cycles 8:85-90 Graham B, Grubbs D, Holland K, Popp B (2007) A rapid ontogenetic shift in the diet of juvenile yellowfin tuna from Hawaii. Mar Biol 150:647-658 Granadeiro JP, Monteiro LR, Furness RW (1998) Diet and feeding ecology of Corys shearwater Calonectris dimomedea in the Azores, north-east Atlantic. Mar Ecol Prog Ser 166: 267-276 Gruber N, Sarmiento N (1997) Global patterns of marine nitrogen fixation and denitrification. Global Biogeochem Cycles11:235266 Hatase H, Takai N, Matsuzawa Y, Sakamoto W, Omuta K, Goto K, Arai N, Fujiwara T (2002) Size-related differences in feeding habitat use of adult female loggerhead turtles Caretta caretta around Japan determined by stable isotope analyses and satellite telemetry. Mar Ecol Prog Ser 233:273-281 Hobson KA, Welch HE (1992) Determination of trophic relationships within a high Arctic marine food web using !13C and !15N analysis. Mar Ecol Prog Ser 84:9-18 Hobson KA, Piatt JF, Pitocchelli J (1994) Using stable isotope to determine seabird trophic relationships. J Anim Ecol 63:786 -789
46 Hobson KA, Schell DM, Renouf D, Noseworthy E (1996) Stable carbon and nitrogen isotopic fractionation between diet and tissues of captive seals: Implications for dietary reconstructions involving marine mammals. Can J Fish and Aquat Sci 53:528533 Huckstadt LA, Rojas CP, Antezana T (2007) Stable isotope analysis reveals pelagic foraging by the Southern sea lion in central Chile. J Exp Mar Biol Ecol 347:123-133 Huskin L, Anadn R, Medina G, Head RN, Harris RP (2001) Mesozooplankton distribution and copepod grazing in the subtropical Atlantic near the Azores: influence of mesoscale structures. J Plankton Res 23:671 -691 Ishibashi H, Daz-Fernndez R, Carrillo E, Koike H (2000) !15N and !13C measurements from the hawksbill turtle, Eretmochelys imbricata, used for scute sourcing. Bulletin of the Graduate School of Social and Cultural Studies, Kyushu University 6:37-45 Kamezaki N, Matsui M (1997) Allometry in the loggerhead turtle, Caretta Caretta. Chelonian Conserv Biol 2:421-425 Le Traon PY, De Mey P (1994) The eddy field associated with the Azores Front east of the Mid -Atlantic Ridge as observed by the Geosat altimeter. J Geophys Res 99:99079923 Limpus CJ, Limpus DJ (2003) The biology of the loggerhead turtle in western south Pacific Ocean foraging areas In: Bolten AB, Witherington BE (eds) Loggerhead Sea Turtles. Smithsonian Books, Washington, DC, p 93113 Liu K, Su M, Hsueh C, Gong G (1996) The nitrogen isotopic composition of nitrate in the Kuroshio Water northeast of Taiwan: Evidence for nitrogen fixation as a source of isotopically light nitrate. Mar Chem 54:273-292 Lochte K, Pfannkuche O (1987) Cyclonic cold-core eddy in the eastern North Atlantic. II. Nutrients, phytoplankton and bacterioplankton. Mar Ecol Prog Ser 39:153-164 Marcoux M, Whitehead H, Rendell L (2007) Sperm whale feeding variation by location, year, social group and clan: evidence from stable isotopes. Mar Ecol Prog Ser 333:309-314 Maros A, Louveaux A, Lelarge C, Girondot M (2006) Evidence of the exploitation of marine resource by the terrestrial insect Scapteriscus didactylus through stable isotope analyzes of its cuticle. BMC Ecol 6:6 Martnez del Rio C, Wolf BO (2005) Mass balance models for animal isotopic ecology. In: Starck MA, Wang T (eds) Physiological and ecological adaptations to feeding in vertebrates. Science Publishers, Enfield, p 141174
47 Martnez del Rio C, Wolf N, Carleton SA, Gannes LZ (2009) Isotopic ecology ten years after a call for more laboratory experiments. Biol Rev 84:91-111 Marzloff M, Shin YJ, Tam J, Travers M, Bertrand A (2009) Trophic structure of the Peruvian marine ecosystem in 2000-2006: Insights on the effects of management scenarios for the hake fishery using the IBM trophic model Osmose. J Mar Sys 75:290-304 Mendes S, Newton J, Reid R, Zuur A, Pierce G (2007) Stable carbon and nitrogen isotope ratio profiling of sperm whale teeth reveals ontogenetic movements and trophic ecology. Oecologia 151:605-615 Minami H, Ogi H (1997) Determination of migratory dynamics of the sooty shearwater in the Pacific using stable carbon and nitrogen isotope analysis. Mar Ecol Prog Ser 158:249256 Minagawa W, Wada E (1984) Stepwise enrichment of 15N along food chains: Further evidence and the relation between !15N and animal age. Geochim Cosmochim Acta 48:1135-1140 Minagawa M, Wada E (1986) Nitrogen isotope ratios of red tide organisms in the East China sea: a characterization of biological nitrogen fixation. Mar Chem 19:2 45-259 Montoya JP, McCarthy JJ (1995) Isotopic fractionation during nitrate uptake by phytoplankton grown in continuous culture. J Plankton Res 17:439464 Montoya JP, Carpenter EJ, Capone DG (2002) Nitrogen fixation and nitrogen isotope abundances in zooplankton of the oligotrophic North Atlantic. Limnol Oceanogr 47:16171628 Montoya JP (2007) Natural abundace of 15N in the marine environment. In: Michener RH, Lajtha K (eds) Stable Isotopes in Ecology and Environmental Science. Wiley Blackwell Publishing, Malden, MA p 176-201 Mourio B, Fernndez E, Etienne H, Hernndez E, Giraud S (2003) Significance of cyclonic subtropical oceanic rings of magnitude(STORM) eddies for the carbon budget of the euphotic layer in the subtropical northeast Atlantic. J Geophys Res 108(C12), 3383, doi:10.1029/2003JC001884 Needoba JA, Waser NA, Harrison PJ, Calvert SE (2003) Nitrogen isotope fractionation in 12 species of marine phytoplankton during growth on nitrate. Mar Ecol Prog Ser 255:81-91 Newsome SD, Martnez del Rio C, Phillips DL, Bearhop S (2007) A niche for isotopic ecology. Front Ecol Environ 5:429436
48 Newsome SD, Tinker MT, Monson DH, Oftedal OT, Ralls K, Staedler MM, Fogel ML, Estes JA (2009) Using stable isotopes to investigate individual diet specialization i n California sea otters (Enhydra lutris nereis) Ecology 90:961 -974 Oschlies A, Garon V (1998) Eddy-induced enhancement of primary production in a model of the North Atlantic Ocean. Nature 394:266-269 Parker DM, Cooke WJ, Balazs GH (2005) Diet of oceanic loggerhead sea turtles (Caretta caretta) in the central North Pacific. Fish Bull 103:142-152 Polovina J, Howell E, Parker D, Balazs G (2003) Dive-depth distribution of loggerhead (Carretta carretta) and olive ridley (Lepidochelys olivacea) sea turtles in the central North Pacific: Might deep longline sets catch fewer turtles? Fish Bull 101:189-193 Popp BN, Laws EA, Bidigare RR, Dore JE, Hanson KL, Wakeham SG (1998) Effect of phytoplankton cell geometry on carbon isotopic fractionation. Geochim Cosmochim Acta 62:69 -77 Popp BN, Graham BS, Olson RJ, Hannides CCS, Lott M, Lpez-Ibarra G, GalvnMagaa G, Fry B (2007) Insight into the trophic ecology of yellowfin tuna, Thunnus albacares, from compound-specific nitrogen isotope analysis of proteinaceous amino acids. In: Dawson T, Siegwolf R (eds) Stable isotopes as indicators of ecological change. Elsevier/Academic, Terrestrial Ecology Series, Amsterdam, p 168184 Post D (2002) Using stable isotopes to estimate trophic position: Models, methods, and assumptions. Ecology 83:703 -718 R Software Program. http://www.r -project.org/ Ramos R, Gonzles-Sols J, Forero MG, Moreno R, Gmez-Daz E, Ruiz X, Hobson KA (2009) The influence of breeding colony and sex on mercury, selenium and lead levels and carbon and nitrogen stable isotope signatures in summer and winter feathers of Calocnectris shearwaters. Oecologia 159:345-354 Rau GH, Sweeney RE, Kaplan IR (1982) Plankton 13C:12C ratio changes with latitude: differences between northern and southern oceans. Deep-Sea Res I 29:1035 -1039 Rau GH, Heyraud M, Cherry RD (1989) 15N/14N and 13C/12C in mesopelagic shrimp from the northeast Atlantic Ocean: evidence for differences in diet. Deep-Sea Res I 36:11031110 Reich K, Bjorndal K, del Rio C (2008) Effects of growth and tissue type on the kinetics of C -13 and N -15 incorporation in a rapidly growing ectotherm. Oecologia 155:651663
49 Revelles M, Cardona L, Aguilar A, Borrell A, Fernandez G, San Felix M (2007) Stable C and N isotope concentration in several tissues of the loggerhead sea turtle Caretta caretta from the western Mediterranean and dietary implications. Sci Mar 71:87-93 Robinson R, Mix A, Martinez P (2007) Southern Ocean control on the extent of denitrification in the southeast Pacific over the last 70 ka. Quat Sci Rev 26:201-212 Rubenstein D, Hobson K (2004) From birds to butterflies: animal movement patterns and stable isotopes. Trends Ecol Evol19:256-263 Saino R, Hattori A (1987) Geographical variation of the water column distribution of suspended particulate organic nitrogen and its 15N natural abundance in the Pacific and its marginal seas. Deep-Sea Res I 34:807 -827 Sanchez RC, Zuta S (2000) The 1997-98 El Nio and its effects on the coastal marine ecosystem off Peru. CalCOFI Rep. 41:62-86 Santos M, Bolten AB, Martins HR, Goncalves J, Riewald B, Bjorndal K (2008) Diving behavior and movements of oceanic stage North Atlantic loggerheads. In: Rees AF, Frick M, Panagopoulou A, Williams K (eds) Proceedings of the Twenty-Seventh Annual Symposium on Sea Turtle Biology and Conservation. NOAA Technical Memorandum NMFS-SEFSC-569 p 58 Shillinger GL, Palacios DM, Bailey H, Bograd SJ, Swithenbank AM, Gaspar P, Wallace BP, Spotila JR, Paladino FV, Piedra R, Eckert SA, Block BA (2008) Persistent leatherback turtle migrations present opportunities for conservation. PLoS Biol 6(7): e171 doi:10.1371/journal.pbio.0060171 Suryan RM, Saba VS, Wallace BP, Hatch SA, Frederiksen M, Wanless S (2009) Environmental forcing on life history strategies: Evidence for multi-trophic level responses at ocean basin scales. Prog Oceanogr 81:214 -222 Takai N, Onaka S, Ikeda Y, Yatsu A, Kidokoro H, Sakamoto W (2000) Geographical variations in carbon and nitrogen stable isotopes in squid. J Mar Biol Assn UK 80:675-684 Tamelander T, Kivime C, Belerby RGJ, Renaud PE, Kristiansen S (2009) Base-line variations in stable isotope values in an Artic marine ecosystem: effects of carbon and nitrogen uptake by phytoplankton. Hydrobiologia 630:6373 Tarazona J, Gutirrez D, Paredes C, Indacochea A (2003) Overview and challenges of marine biodiversity research in Peru. Gayana 67(2):206-231 Vaillancourt RD, Marra J, Seki MP, Parson ML, Bigidare RR (2003) Impact of a cyclonic eddy on phytoplankton community structure and photosynthetic competency in the subtropical North Pacific Ocean. Deep-Sea Res I 50:829-847
50 Voss M, Dippner JW, Montoya JP (2001) Nitrogen isotope patterns in the oxygen deficient waters of the Eastern Tropical North Pacific Ocean. Deep-Sea Res I 48:1905-1921 Wada E, Hattori A (1976) Natural abundance of 15N in particulate organic matter in the North Pacific Ocean. Geochim Cosmochim Acta 40:249-251 Wada E, Hattori A (1991) Nitrogen in the sea: forms, abundances, and rate processes. CRC, FL, USA Wallace B, Seminoff J, Kilham S, Spotila J, Dutton P (2006) Leatherback turtles as oceanographic indicators: stable isotope analyses reveal a trophic dichotomy between ocean basins. Mar Biol 149:953960 Wang C, Fiedler P (2006) ENSO variability and the eastern tropical Pacific: A review. Prog Oceanogr 69:239-266 Ward BB, Devol AH, Rich JJ, Chang BX, Bulow SE, Naik H, Pratihary A, Jayakumar A (2009) Denitrification as the dominant nitrogen loss process in the Arabian Sea. Nature 461:78-81 Wu J, Calvert SE, Wong CS (1997) Nitrogen isotope variations in the subarctic northeast Pacific: relationships to nitrogen utilization and trophic structure. Deep-Sea Res I 44:287-314
51 BIOGRAPHICAL SKETCH Mariela Pajuelo was born in Lima, Peru. She is one of four children of Fortunato Pajuelo Matos and Ricardina Rubina Salazar. She attended Universidad Nacional Mayor de San Marcos in Lima, Peru, and obtained her bachelors degree in biology with emphasis on hydrobiology and fisheries science. Mariela joined Peruvian, non governmental organization Pro Delphinus in 2004 and has been involved in marine conservation ever since. She was assigned her first sea turtle project in 2005, closely working with fishermen, while testing new mitigation measures to avoid incidental capture of sea turtles. She joined the Department of Zoolog y at the University of FloridaGainesville in 2007 and started working toward her masters degree under the supervision of Dr. Karen A. Bjorndal, looking at the trophic ecology of oceanic juvenile loggerhead turtles. She completed her Master of Science degree in 2010.