An otolith microchemistry study of possible relationships between the origins of leptocephali of European eels in the Sargasso Sea and the continental destinations and relative migration

An otolith microchemistry study of possiblerelationships between the origins of leptocephaliof European eels in the Sargasso Sea and thecontinental destinations and relative migrationsuccess of glass eels

Introduction

Despite the intensive research dedicated to Atlanticeels, little is known about the extent to which eelscoming from different European rivers converge onthe same spawning site (Daemen et al. 2001; Maes &Volckaert 2002; Van Ginneken et al. 2005). Based oncatches of leptocephali, the European eel wouldputatively spawn from March to June in the SargassoSea within a large ellipse which extends east-west

between 48 and 74�W longitude and south-northbetween 23 and 30�N latitude (Boetius & Harding1985; Kleckner & Mccleave 1988; Mccleave 2003).

The distribution of small leptocephali in relation tothe hydrographic structure in the southern SargassoSea showed that spawning occurred south of a distincttemperature front in the Subtropical ConvergenceZone (STCZ) of the Sargasso Sea (Kleckner &Mccleave 1988). This temperature front appeared toform at the same water density each year during the

Ecology of Freshwater Fish 2010: 19: 627–637Printed in Malaysia Æ All rights reserved

� 2010 John Wiley & Sons A/S

ECOLOGY OFFRESHWATER FISH

Martin J, Daverat F, Pecheyran C, Als TD, Feunteun E, Reveillac E. Anotolith microchemistry study of possible relationships between the originsof leptocephali of European eels in the Sargasso Sea and the continentaldestinations and relative migration success of glass eels.Ecology of Freshwater Fish 2010: 19: 627–637. � 2010 John Wiley &Sons A ⁄S

Abstract – Little is known about the extent to which Atlantic eels comingfrom different European rivers converge on the same spawning site. Ouraim was to evaluate the spatial homogeneity of eel spawning area(s) withan otolith microchemistry approach. This work compared the elementalsignatures of otolith’s core region of Anguilla anguilla leptocephali caughtin the Sargasso Sea in 2007 with those of glass eels and elvers sampled inEuropean estuaries during 2006, 2008 and 2009. Using laser ablationinductively coupled plasma mass spectrometry, the same annular ablationtrajectory along the first feeding mark was applied on otoliths of glass eels,elvers and leptocephali. The concentrations of thirteen isotopes in theotoliths of glass eels ⁄ elvers did not vary among three annual cohortscollected in eleven European locations. However, otolith elementalfingerprints of leptocephali differed significantly from glass eels otolith’ssignature. Although the mechanisms that regulate the differences in traceelement signatures among leptocephali and glass eels ⁄ elvers areunknown, we propose that the sampled glass eels ⁄ elvers were born in aspawning site or region where favourable transport and ⁄or feedingconditions occurred. Conversely, the leptocephali may have been sampledin a less favourable region in the Sargasso Sea, with a low probabilityof reaching continental growth areas.

J. Martin1, F. Daverat1,C. Pecheyran2, T. D. Als3,E. Feunteun4, E. Reveillac4

1Cemagref, Estuarine Ecosystems and Diadro-mous Fish Research Unit, 50 avenue de VerdunF–33612 Cestas Cedex, France, 2Universite dePau et des Pays de l’Adour, UMR 5254 IPREM-LCABIE, 2 avenue Angot, Helioparc Pau Pyrenee,64053 Pau cedex 9, France, 3DTU Aqua, NationalInstitute of Aquatic Resources, Technical Uni-versity of Denmark, Vejlsøvej 39, 8600 Silkeborg,France, 4Museum National d’Histoire Naturelle,UMR 7208 BOREA, CRESCO, 38 rue du PortBlanc, 35800 Dinard, France

Key words: Anguilla anguilla ; otolith micro-chemistry; spawning events; laser ablationinductively coupled plasma mass spectrometry

F. Daverat, Cemagref, Estuarine Ecosystems andDiadromous Fish Research Unit, 50 avenue deVerdun F-33612 Cestas Cedex, France;e-mail: [email protected]

Accepted for publication July 5, 2010

doi: 10.1111/j.1600-0633.2010.00444.x 627

spawning season (Miller & Mccleave 1994) andseemed to act as a boundary for many organisms.Features of this frontal zone, such as odour ortemperature, may serve as cues to silver eels to ceasemigration and spawn. Whether the spawning area islocated in a single site or at distinct sites is stillunknown. The European eel spawning area, partlyshared with the American eel, appears much morediffuse than the very localised spawning area of theJapanese eel (Tsukamoto 2006).

Nevertheless, no adult eels have ever been capturedin the Sargasso Sea. Tracking of European silver eelmigration as part of the EELIAD project (http://www.eeliad.com) suggests a route against Atlanticdrift currents and toward the Canary and AzoresCurrent systems (Aarestrup et al. 2009). Surprisingly,the mean horizontal net migration speed (13.8 kmÆday)1) was much lower than required (35 kmÆday)1)to reach the Sargasso Sea for spawning as expected inspring. Furthermore, another major source of contro-versy is the timing of migration of eel larvae from thespawning area(s) to the coastal areas. Lagrangiantracks of leptocephalus drift on modelled circulationfields showed that the most rapid transport of larvae tonear Europe was about 2 years (Bonhommeau et al.2009b; Kettle & Haines 2006) while the otolithincrements counts gave less than a year betweenhatching and metamorphosis onset (Arai et al. 2000;Lecomte Finiger 1994). Bonhommeau et al. (2009b)estimated that larval mortality was potentially veryhigh and increased markedly with duration of theoceanic migration. Mortality might also be higher andhence migration success lower for larvae originatingfrom spawning events in certain areas of the SargassoSea, according to Kettle & Haines (2006) andBonhommeau et al. (2008).

A technique that could be used to clarify possiblelinks between larval origins in different areas of theSargasso, migrations to different continental growthhabitats and return to spawning areas by silver eels isotolith ‘chemical fingerprinting’. Recent studies haveshown that the elemental composition of fish otolithsmay be a powerful means for distinguishing amongmovements and life histories of fish stocks (Elsdonet al. 2008). First, otoliths grow throughout the life ofthe fish and are considered chemically inert (Campana& Neilson 1985). Second, the calcium carbonate andtrace elements that make up 90% of the otolith appear tobe mainly derived from water (Campana et al. 1994).Therefore, the composition of the otolith core region,more precisely layers incremented just after first feedingoccurs, when the larva had completely utilised its yolkreserves, is expected to reflect the composition of thewater of the spawning site. Thus, fishes spawned atdifferent sites, with different water chemical composi-tions, would exhibit different chemical fingerprints of

their otolith core region (Campana et al. 1994). Con-versely, if there is a unique spawning area for all eels, itis expected that the chemical fingerprint will be thesame for all individuals’ otoliths.

To test these alternatives, we compared the elemen-tal signatures of otolith’s core region of Anguillaanguilla leptocephali caught in the Sargasso Sea inApril 2007 with those of glass eels and elvers sampledat their arrival in eleven European estuaries during3 years (2006, 2008 and 2009). Those findings mightbe of use in informing management and conservationof the European eel in the light of declines inrecruitment in recent decades (ICES 2008). This studywas carried out as part of the EELIAD project, themain aims of which were to identify migrationpathways and spawning areas of European silver eelsand clarify whether panmixia occurs. Leptocephaliwere provided from catches made during the Galathea3 Cruise (http://www.galathea.dk).

Materials and methods

Sampling

More than 300 specimens of A. anguilla glass eels andelvers were collected in eleven locations across Europeduring 2006, 2008 and 2009: W and SWAtlantic coast(France), Mediterranean lagoon (France), SW Eng-land, W coast of Sweden, the Netherlands, Spain andIreland (Table 1, Fig. 1). Sixteen A. anguilla lepto-cephali, with sizes (total length) ranging from 12 to15 mm captured in the Sargasso Sea in April 2007were also included in the analysis (Table 2; Fig. 2).These were collected during the Danish Galathea 3expedition in March and April 2007. Thirty-threestations distributed along three transects at longitudes64�W, 67�W and 70�W were sampled by the vessel

Table 1. Location of Anguilla anguilla glass eel and elver samples takenacross Europe.

Samplingstation Country Sampling date

Lifestage N

Severn England Spring 2008 G 45Canet France 12 ⁄ 01 ⁄ 2008

and 23 ⁄ 01 ⁄ 2008G 45

Gironde France 4 ⁄ 16 ⁄ 2008 G 45Ringhals Sweden 3 ⁄ 15 ⁄ 2008 G 45Den Oever The Netherlands 2 ⁄ 27 ⁄ 2006 G 19Aiguillon France 2 ⁄ 15 ⁄ 2006 G 22Oria France 2 ⁄ 27 ⁄ 2006 G 19Stes Maries

de la MerFrance 2 ⁄ 15 ⁄ 2006 G 21

Burrishoole Ireland 04 ⁄ 06 ⁄ 2008and 24 ⁄ 06 ⁄ 2008

E 9

Erne Ireland Spring 2008 E 9Carlingford Ireland 3 ⁄ 12 ⁄ 2009 G 28

N, number of samples; G, Glass eels; E, Elvers.

Martin et al.

628

‘Vaedderen’, and an additional four stations weresampled by the accompanying vessel ‘NewfoundlandAlert’ (Fig. 2). Fish larvae were sampled using a ringnet (3.5 m diameter) equipped with a 25 m long netwith 600 lm mesh. This gear was lowered to 250 m inan oblique haul at a speed of 2.5 knots and a wire pay-out and retrieval of 25 mÆmin)1 and 15 mÆmin)1,respectively. The catch was kept cold, and within 1 h,Anguilla-like larvae were identified, photographed,total length measured and preserved in RNAlater�(Qiagen, Hilden, Germany). The remaining samplewas preserved in 96% ethanol for the identification ofadditional Anguilla-like larvae. RNAlater dissolvesotoliths and otoliths were therefore only available forlarvae stored in ethanol, leaving only a small sampleof larvae with a limited geographical distributionavailable for analysis of otolith chemistry (Table 2). Itwas verified with genetic tools that the 16 leptocephaliwere A. Anguilla.

Otolith preparation

Otolith pairs (Sagitta) were extracted from each eel’shead, preserved in 70% ethanol, washed in ultra pure(milliQ-water) and then dried at 40 �C for 24 h. OneSagitta per fish was embedded in epoxy resin with theprimordial surface downwards. Resin blocks wereground with ultrapure water and sandpaper (1200–4000 grit) until the primordium was reached. Finally,otoliths were rinsed with ultra pure-water, and then air-dried before being stored in individually labelledplastic vials. During all sample preparation, samplesonly came in contact with nonmetallic and acid-leached equipment.

Laser ablation

A high repetition rate Infra Red femtosecond laserablation system (Alfamet, Novalase SA – AmplitudeSystemes, France) was used in this study. It delivers360 fs pulses at a wavelength of 1030 nm and can beoperated at repetition rates from 1 Hz to 10 kHz.A galvanometric scanning beam device fitted betweenthe laser source and the objective (25 mm focallength) permits the user to move (up to 280 lmÆs)1) asmall laser beam with a high repositioning precision(<1 lm). When operated at high repetition rate, thisunique system allows ablating the sample very rapidlyaccording to two-dimensional trajectories as reportedelsewhere (Ballihaut et al. 2007; Barats et al. 2007a;Fernandez et al. 2007). These abilities were fullyexploited to obtain the best signal sensitivity whilekeeping the best spatial resolution in three dimensionson the otoliths. Special care was also taken in order toprevent excessive depth penetration of the laser beamthat would likely induce confusion between aragonitelayers. Crater depth was 15 lm.

An annular ablation trajectory (15 lm ± 1 awayfrom the primordium), along the first feeding mark(Lecomte-Finiger & Yahyaoui 1989) was applied onotoliths of glass eels, elvers and leptocephali, whichavoided the maternal influence (primordium) on thechemical signature (Fig. 3) (Kalish 1990; Riemanet al. 1994). We assume that the first feeding mark wasalways at 15 lm from the primordium for each sampleof glass eels, elvers and leptocephali (Wang & Tzeng2000). The ablation pit size used in this study was adiameter of 10 lm and samples were fired using a20 mW energy pulses.

Fig. 1. Map of the glass eels and elvers sampling locations inEurope.

Table 2. Location of Anguilla anguilla leptocephali samples captured in the Sargasso Sea.

Stations Numbers of leptocephali Start date End date Start position End position

NA20 3 4 ⁄ 11 ⁄ 2007 4 ⁄ 11 ⁄ 2007 26�09¢N; 64�86¢W 26�16¢N; 64�97¢WNA21 10 4 ⁄ 11 ⁄ 2007 4 ⁄ 12 ⁄ 2007 26�16¢N; 64�98¢W 26�10¢N; 64�88¢WNA22 3 4 ⁄ 12 ⁄ 2007 4 ⁄ 12 ⁄ 2007 26�10¢N; 64�87¢W 26�20¢N; 65�01¢W

Evaluate the homogeneity of eel spawning area with an otolith microchemistry

629

The femtosecond laser system was coupled to anElan DRC2 ICPMS (Perkin Elmer SCIEX InductivelyCoupled PlasmaMass Spectrometer, Concord, Ontario,Canada) via a dual sample introduction device used tomix the dry aerosol generated by the laser with a wetaerosol produced by a pneumatic nebulisation. Thisconfiguration was found to prevent carbonate depositson the torch injector and to provide robust plasmaconditions (Barats et al. 2007b; Thebault et al. 2009).

The concentrations of thirteen isotopes, representingten elements, were monitored in the core region ofeach sample (24Mg, 25Mg, 63Cu, 65Cu, 43Ca, 55Mn,68Zn, 66Zn, 85Rb, 59Co, 7Li, 86Sr and 138Ba). Details ofthe operating conditions are given in Table 3.

Calcium was used as an internal standard toimprove the reliability of the concentration measure-ment (Campana 1999). The calcium concentration was

normalised to be 400,000 lgÆg)1 based on the stoi-chiometry of calcium carbonate, and the concentra-tions of other elements were calculated by internalcorrection using Ca. Quantification of trace elementsin otoliths was achieved by external calibration using aseries of 13 co-precipitated carbonate pellets (Baratset al. 2007b) ranging from 0.1 to 500 lgÆg)1. Thecalibration curve was systematically repeated after 2 hworking using three enriched pellets and the CRMNIES 22. This Certified Reference Material, producedby the National Institute for Environmental Studies, isintended for use in the quality assurance of theanalysis of selected major, minor and trace elements infish otolith and similar marine aragonite matrices.

Fig. 2. Map showing the collection sites ofAnguilla anguilla leptocephali in the Sar-gasso Sea.

Fig. 3. Representative otolith image with an annular ablationtrajectory along the first feeding mark (15 lm away from theprimordium).

Table 3. Operating conditions of the laser ablation inductively coupledplasma mass spectrometry system used in this study.

ICPMSInstrumentation Perkin Elmer DRCIIAcquisition mode Peak JumpingCoolant 15 lÆmin)1

Auxiliary gas flow rate 1.2 lÆmin)1

Carrier gas flow rate 0.68 lÆmin)1 (laser He carrier gas)0.72 lÆmin)1 (Ar make-up gas)

Dwell timeMonitored isotopes 24Mg, 25Mg, 63Cu, 65Cu, 43Ca,

55Mn, 68Zn, 66Zn, 85Rb, 59Co,7Li, 86Sr, 138Ba

Total acquisition time 20 s per run

Laser ablationInstrumentation ALFAMET (novalase Sa)Laser type Femtosecond KGW doped with YbWavelength 1030 nmPulse duration 360 fsLaser pulse energy 40 lJÆpulse)1

Repetition rate 500 HzBeam size (1 ⁄ e2 criterion) 10 lmLaser beam velocity 1 mmÆs)1

ICPMS, inductively coupled plasma mass spectrometry.

Martin et al.

630

A series of the samples were arranged randomlyinto the ablation cell to avoid the possibility ofconfounding potential instrument drift effects withstock differences. The limits of detection (lgÆg)1)achieved in this study were as follows: 24Mg 3.2–5.6,25Mg 2.5–7, 63Cu 0.2–5, 65Cu 0.3–6, 43Ca 109–495,55Mn 0.3–3.4, 68Zn 0.7–2.2, 66Zn 0.6–2, 85Rb 0.1–0.4,59Co 0.11.2, 7Li 0.2–0.5, 86Sr 4.2–6.3 and 138Ba 0.05–0.3. They were based on a 3r criterion, where r is thestandard deviation for the mean blank count of eachisotope. All the element concentrations in the otolithwere above the detection limits. All elemental datawere expressed as molar ratios to Ca.

Statistical analyses

As the assumptions of homogeneity of variances andof multivariate normal distribution for each element ateach site were not met, differences among sites perelement were tested using the nonparametric Mann–Whitney U ⁄Wilcoxon tests with a Bonferroni adjust-ment. Lachenbruch (1975) indicates that discriminantanalysis is relatively robust even when there aremodest violations of the assumptions of homogeneityof variances and multivariate normal distribution(Klecka 1980). Therefore, a canonical discriminantanalysis (CDA) was used to determine if overallelemental ratio at the first feeding mark differedbetween sampled sites and a quadratic discriminantfunction was used to classify the samples. Finally,classification success of each of the sampled sites wasdetermined through jackknifed discriminant analysis.For all the statistical analysis, levels of significancewere 5%.

Results

A total of 13 isotopes representing ten elements werefound in detectable concentrations in glass eels, elversand leptocephali otoliths using Laser Ablation Induc-tively Coupled Plasma Mass Spectrometry (LA-IC-PMS). A total of 307 glass eels and elvers collectedfrom 11 sites in 2006, 2008, 2009 and 16 leptocephalifrom the Sargasso Sea (2007) were examined for theseelements at their otolith first feeding mark. Theisotopes presenting the highest ratios were 86Sr,24Mg and 25Mg (Table 4).

For the glass eel ⁄ elvers group, the nonparametricMann–Whitney U ⁄Wilcoxon tests revealed that themean element ratios of 24Mg, 63Cu, 65Cu, 55Mn, 66Zn,85Rb, 59Co, 7Li, 86Sr, 138Ba of individual eel otolithsvaried significantly between sites (P < 0.05), but nosignificant differences were observed for 68Zn:Ca and25Mg:Ca ratios (Table 5). Pairwise comparisons indi-cated that the Saintes Maries de la Mer site oftendiffered significantly from Ringhals, Severn, Gironde

Table 4. Mean ratio ± SE of 12 isotopes at the first feeding mark region ofotoliths of Anguilla anguilla glass eels ⁄ elvers collected at several sites during2006, 2008, 2009 and leptocephali collected in the Sargasso Sea during2007. Glass eels and elvers are represented by their capture locations (seeTable 1) and leptocephali are stated as ‘lepto.’ in the legend.

Isotope SiteIsotope ⁄ Ca ratios,mean ± SE Range

Mn55 Lepto. 1.66E-05 ± 3.10E-06 5.00E-06–5.80E-05Burrishoole 7.89E-06 ± 1.59E-06 2.81E-06–1.79E-05Canet 5.89E-06 ± 4.61E-07 1.39E-06–1.41E-05Carlingford 8.86E-06 ± 7.54E-07 2.42E-06–2.19E-05Denoever 6.57E-06 ± 5.50E-07 2.35E-06–1.04E-05Erne 7.16E-06 ± 1.92E-06 3.99E-07–2.12E-05Gironde 6.58E-06 ± 4.72E-07 0–1.78E-05Aiguillon 9.22E-06 ± 1.19E-06 2.79E-06–2.75E-05Oria 6.44E-06 ± 6.89E-07 1.43E-06–1.16E-05Ringhals 5.25E-06 ± 4.13E-07 1.42E-06–1.70E-05Severn 5.34E-06 ± 4.52E-07 8.53E-07–1.35E-05SteMaries 8.07E-06 ± 5.51E-07 2.20E-06–1.29E-05

Sr86 Lepto. 1.12E-02 ± 3.85E-04 8.33E-03–1.40E-02Burrishoole 1.19E-02 ± 5.52E-04 8.21E-03–1.36E-02Canet 1.07E-02 ± 3.30E-04 5.62E-03–1.44E-02Carlingford 1.15E-02 ± 3.02E-04 7.09E-03–1.39E-02Denoever 1.45E-02 ± 4.59E-04 1.22E-02–1.93E-02Erne 1.18E-02 ± 3.12E-04 1.04E-02–1.37E-02Gironde 1.11E-02 ± 2.46E-04 6.60E-03–1.44E-02Aiguillon 1.45E-02 ± 3.47E-04 1.14E-02–1.80E-02Oria 1.28E-02 ± 2.87E-04 1.06E-02–1.54E-02Ringhals 1.10E-02 ± 2.05E-04 6.37E-03–1.39E-02Severn 1.17E-02 ± 2.60E-04 7.35E-03–1.53E-02SteMaries 1.23E-02 ± 3.24E-04 9.57E-03–1.57E-02

Ba138 Lepto. 1.38E-05 ± 5.06E-06 3.08E-06–8.67E-05Burrishoole 2.99E-06 ± 2.05E-07 2.10E-06–3.94E-06Canet 4.23E-06 ± 2.61E-07 2.52E-06–1.17E-05Carlingford 3.53E-06 ± 1.92E-07 2.05E-06–6.89E-06Denoever 5.49E-06 ± 2.45E-07 3.30E-06–7.52E-06Erne 3.43E-06 ± 2.43E-07 2.60E-06–4.55E-06Gironde 4.26E-06 ± 1.76E-07 2.47E-06–6.71E-06Aiguillon 5.37E-06 ± 3.31E-07 3.68E-06–1.02E-05Oria 5.76E-06 ± 3.04E-07 4.35E-06–8.69E-06Ringhals 3.94E-06 ± 1.47E-07 2.60E-06–7.62E-06Severn 4.07E-06 ± 2.08E-07 2.41E-06–1.11E-05SteMaries 4.25E-06 ± 1.93E-07 2.23E-06–5.82E-06

Zn68 Lepto. 1.26E-05 ± 2.90E6 0–3.77E-05Burrishoole 3.56E-06 ± 1.16E-06 0–9.26E-06Canet 9.78E-06 ± 1.35E-06 0–3.45E-05Carlingford 4.59E-06 ± 8.55E-07 0–2.33E-05Denoever 7.07E-06 ± 1.66E-06 0–2.84E-05Erne 3.23E-06 ± 4.55E-07 1.90E-06–5.52E-06Gironde 8.50E-06 ± 1.23E-06 0–3.58E-05Aiguillon 7.61E-06 ± 1.67E-06 2.18E-07–3.01E-05Oria 6.11E-06 ± 1.31E-06 0–2.29E-05Ringhals 4.49E-06 ± 5.90E-07 0–1.99E-05Severn 7.34E-06 ± 1.03E-06 0–2.63E-05SteMaries 7.41E-06 ± 2.04E-06 0–3.71E-05

Zn66 Lepto. 1.35E-05 ± 3.63E-06 1.64E-06–4.90E-05Burrishoole 2.99E-06 ± 7.74E-07 3.95E-07–7.81E-06Canet 7.72E-06 ± 1.19E-06 1.04E-06–4.48E-05Carlingford 4.00E-06 ± 5.07E-07 0–1.09E-05Denoever 8.49E-06 ± 1.96E-06 1.30E-06–3.54E-05Erne 2.59E-06 ± 1.09E-06 2.58E-07–1.04E-05Gironde 5.38E-06 ± 7.81E-07 0–2.69E-05Aiguillon 4.55E-06 ± 7.33E-07 0–1.54E-05Oria 4.63E-06 ± 1.05E-06 0–1.92E-05Ringhals 5.15E-06 ± 6.55E-07 0–1.95E-05


631

and Canet sites and that the Den Oever site oftendiffered from Canet, Ringhals and Severn (Table 5).Despite the statistical significance, many of thebetween-site differences were relatively small andthere was no evidence for a correlation between‘chemical fingerprints’ versus geographical distances.The year of capture did not account for the inter-sitedifferences in isotope concentration. There was nosignificant variation in isotope concentration amongdates of capture (Friedman’s tests, P > 0.10).

Considering all samples, otolith elemental finger-prints of leptocephali differed significantly from glasseel ⁄ elvers otolith signature (Kruskal–Wallis test,P < 0.05) (Table 5). The mean element ratios of24Mg, 25Mg, 63Cu, 65Cu, 55Mn, 66Zn, 7Li and 138Bain leptocephali otoliths were systematically higherthan in glass eel ⁄ elvers otoliths (P < 0.05) while nosignificant differences were apparent in the 68Zn:Caand 59Co:Ca ratios among glass eels ⁄ elvers andleptocephali (P > 0.05).

Canonical discriminant analysis, based on concen-trations of 11 isotopes, showed that the leptocephalisamples separated clearly from all glass eel ⁄ elversamples along the first canonical variate (Fig. 4). Thesame results were obtained using a cluster analysisbased on Euclidean distances and a farthest-neighbourparadigm. The first two discriminant functionsexplained more than 70% of the variation in the data.Leptocephali had higher values on canonical value(CV) 1, which was related to higher ratios of Li7,Mn55, Mg25 and Mg24 than glass eels and elvers

Table 4. (Continued ).


Severn 3.85E-06 ± 3.71E-07 0–1.00E-05SteMaries 3.12E-06 ± 8.18E-07 0–1.61E-05

Rb85 Lepto. 6.27E-06 ± 1.26E-06 1.43E-06–2.06E-05Burrishoole 3.77E-05 ± 5.20E-06 1.64E-05–6.78E-05Canet 1.93E-06 ± 3.98E-07 0–1.06E-05Carlingford 3.88E-05 ± 3.19E-06 1.22E-05–6.78E-05Denoever 2.12E-05 ± 1.56E-06 8.26E-06–3.35E-05Erne 4.41E-05 ± 6.28E-06 1.75E-05–8.39E-05Gironde 3.54E-06 ± 4.65E-07 4.32E-07–1.57E-05Aiguillon 2.36E-05 ± 1.56E-06 1.26E-05–4.08E-05Oria 1.68E-05 ± 1.08E-06 7.54E-06–2.58E-05Ringhals 1.72E-06 ± 4.07E-07 1.87E-07–1.74E-05Severn 5.79E-06 ± 3.06E-07 1.98E-06–1.33E-05SteMaries 3.51E-06 ± 4.23E-07 1.00E-06–7.79E-06

Co59 Lepto. 1.05E-06 ± 1.08E-07 3.57E-07–2.00E-06Burrishoole 9.37E-07 ± 9.02E-08 5.82E-07–1.35E-06Canet 1.34E-06 ± 5.69E-08 4.94E-07–1.97E-06Carlingford 1.23E-06 ± 6.77E-08 5.34E-07–1.87E-06Denoever 1.32E-06 ± 7.79E-08 7.52E-07–2.17E-06Erne 9.03E-07 ± 1.40E-07 4.92E-07–1.74E-06Gironde 1.28E-06 ± 6.40E-08 6.45E-07–3.19E-06Aiguillon 1.45E-06 ± 9.60E-08 6.86E-07–2.32E-06Oria 1.77E-06 ± 1.24E-07 1.10E-06–3.51E-06Ringhals 1.36E-06 ± 3.83E-08 8.63E-07–2.29E-06Severn 1.11E-06 ± 5.10E-08 3.66E-07–1.80E-06SteMaries 1.64E-06 ± 9.92E-08 8.42E-07–2.50E-06

Li7 Lepto. 2.26E-06 ± 2.10E-07 1.18E-06–4.59E-06Burrishoole 1.00E-06 ± 1.78E-07 2.64E-08–1.72E-06Canet 1.49E-06 ± 7.24E-08 4.32E-07–2.84E-06Carlingford 1.24E-06 ± 9.99E-08 3.24E-07–2.23E-06Denoever 1.40E-06 ± 7.94E-08 9.36E-07–2.19E-06Erne 1.37E-06 ± 2.05E-07 5.02E-07–2.58E-06Gironde 2.01E-06 ± 1.15E-07 8.32E-07–5.36E-06Aiguillon 1.43E-06 ± 9.29E-08 6.44E-07–2.24E-06Oria 1.65E-06 ± 1.14E-07 7.30E-07–2.65E-06Ringhals 1.67E-06 ± 7.11E-08 8.45E-07–2.88E-06Severn 2.23E-06 ± 7.75E-08 1.23E-06–3.52E-06SteMaries 1.60E-06 ± 8.95E-08 8.85E-07–2.33E-06

Mg24 Lepto. 4.83E-04 ± 1.15E-04 1.44E-04–2.00E-03Burrishoole 1.10E-04 ± 1.37E-05 7.69E-05–2.02E-04Canet 9.00E-05 ± 6.74E-06 5.06E-05–3.09E-04Carlingford 1.33E-04 ± 8.06E-06 7.02E-05–2.76E-04Denoever 7.47E-05 ± 4.05E-06 4.20E-05–1.05E-04Erne 9.58E-05 ± 1.21E-05 4.72E-05–1.57E-04Gironde 8.63E-05 ± 3.30E-06 5.06E-05–1.46E-04Aiguillon 8.31E-05 ± 7.82E-06 5.33E-05–2.08E-04Oria 7.57E-05 ± 1.94E-06 5.99E-05–9.40E-05Ringhals 9.04E-05 ± 8.80E-06 3.98E-05–3.24E-04Severn 1.25E-04 ± 3.43E-06 6.28E-05–1.78E-04SteMaries 1.05E-04 ± 9.82E-06 4.50E-05–2.06E-04

Mg25 Lepto. 4.53E-04 ± 1.17E-04 1.20E-04–2.05E-03Burrishoole 7.41E-05 ± 6.19E-06 5.58E-05–1.13E-04Canet 7.69E-05 ± 6.08E-06 4.46E-05–2.81E-04Carlingford 1.16E-04 ± 1.19E-05 5.41E-05–3.70E-04Denoever 7.33E-05 ± 7.58E-06 3.34E-05–1.76E-04Erne 7.31E-05 ± 1.11E-05 3.12E-05–1.19E-04Gironde 7.17E-05 ± 3.11E-06 3.68E-05–1.18E-04Aiguillon 7.44E-05 ± 6.31E-06 3.43E-05–1.60E-04Oria 6.97E-05 ± 5.00E-06 4.77E-05–1.22E-04Ringhals 7.95E-05 ± 8.37E-06 3.09E-05–2.78E-04Severn 1.04E-04 ± 3.54E-06 5.14E-05–1.87E-04SteMaries 9.66E-05 ± 1.08E-05 3.28E-05–1.96E-04

Table 4. (Continued ).


Cu63 Lepto. 1.79E-05 ± 6.81E-06 4.61E-07–1.05E-04Burrishoole 1.57E-06 ± 4.09E-07 0–4.09E-06Canet 6.12E-07 ± 8.81E-08 0–3.21E-06Carlingford 9.91E-07 ± 1.78E-07 0–4.05E-06Denoever 2.63E-06 ± 2.53E-07 1.25E-06–5.33E-06Erne 1.00E-06 ± 3.05E-07 0–2.73E-06Gironde 6.49E-07 ± 8.45E-08 0–2.50E-06Aiguillon 2.28E-06 ± 3.16E-07 6.48E-08–5.03E-06Oria 6.12E-07 ± 1.26E-07 0–1.68E-06Ringhals 6.05E-07 ± 7.87E-08 0–2.97E-06Severn 1.81E-07 ± 1.68E-08 0–5.04E-07SteMaries 9.74E-07 ± 2.21E-07 7.14E-08–4.35E-06

Cu65 Lepto. 1.71E-05 ± 5.86E-06 2.69E-07–8.37E-05Burrishoole 5.64E-06 ± 1.89E-06 0–1.69E-05Canet 1.24E-06 ± 1.29E-07 0–3.49E-06Carlingford 3.27E-06 ± 6.67E-07 0–1.28E-05Denoever 4.31E-06 ± 2.98E-07 2.27E-06–6.57E-06Erne 2.30E-06 ± 6.53E-07 0–5.43E-06Gironde 1.31E-06 ± 1.68E-07 0–5.31E-06Aiguillon 3.71E-06 ± 3.46E-07 1.02E-06–8.25E-06Oria 1.51E-06 ± 2.46E-07 3.38E-07–4.02E-06Ringhals 1.41E-06 ± 1.48E-07 2.08E-07–4.38E-06Severn 3.65E-07 ± 3.25E-08 0–9.47E-07SteMaries 2.21E-06 ± 3.77E-07 4.33E-07–6.09E-06

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Table 5. Mean otolith isotopic ratios, adjusted forCa43, by sample site. Isotopes that differedsignificantly (P < 0.05) between sites are markedwith an asterisk. Where present, significant pair-wise differences with another site (represented byits first letter) are indicated in parentheses. Glasseels and elvers are represented by their capturelocations (see Table 1) and leptocephali are statedas ‘lepto.’ in the legend.

Isotope Lepto. Severn

Ba138* 1.38E-05 (A,B,Can,Car,D,E,G,O,R,S,Ste) 4.07E-06 (D,L)Zn68 1.26E-05 7.34E-06Zn66* 1.35E-05 (A,G,O,R,S,Ste) 3.85E-06 (L)Rb85* 6.27E-06 (Can) 5.79E-06 (Can,O,R,Ste)Li7* 2.26E-06 (A,B,Can,Car,D,E,G,O,R,S,Ste) 2.23E-06 (B,Can,Car,L,R)Mg24* 4.83E-04 (A,B,Can,Car,D,E,G,O,R,S,Ste) 1.25E-04 (Can,E,G,L,O,R)Mg25* 4.53E-04 (A,B,Can,Car,D,E,G,O,R,S,Ste) 1.04E-04 (L)Mn55* 1.66E-05 (A,B,Can,Car,D,E,G,O,R,S,Ste) 5.34E-06 (L)Cu63* 1.79E-05 (A,B,Can,Car,D,E,G,O,R,S,Ste) 1.81E-07 (D,L)Cu65* 1.71E-05 (A,Can,Car,G,O,R,S,Ste) 3.65E-07 (A,D,L)Co59* 1.05E-06 1.11E-06 (Ste)Sr86* 1.12E-02 (Car,E) 1.17E-02 (B,Car,E,Ste)

Isotope Gironde Canet

Ba138* 4.26E-06 (B,L,R) 4.23E-06 (L)Zn68 8.50E-06 9.78E-06Zn66* 5.38E-06 (L) 7.72E-06 (Ste)Rb85* 3.54E-06 (Can,O,Ste) 1.93E-06 (A,Car,D,E,G,L,O,R,S)Li7* 2.01E-06 (L,R) 1.49E-06 (L,S,Ste)Mg24* 8.63E-05 (Can,L,S) 9.00E-05 (G,L,S)Mg25* 7.17E-05 (L) 7.69E-05 (L)Mn55* 6.58E-06 (L) 5.89E-06 (L)Cu63* 6.49E-07 (D,L) 6.12E-07 (A,D,L)Cu65* 1.31E-06 (A,D,L) 1.24E-06 (A,D,L)Co59* 1.28E-06 (Ste) 1.34E-06 (Ste)Sr86* 1.11E-02 (B,Car,E,O,Ste) 1.07E-02 (B,Car,E,Ste)

Isotope Ringhals Aiguillon

Ba138* 3.94E-06 (D,G,L) 5.37E-06 (B,L)Zn68 4.49E-06 7.61E-06Zn66* 5.15E-06 (L) 4.55E-06 (Car,L)Rb85* 1.72E-06 (Can,S) 2.36E-05 (Can)Li7* 1.67E-06 (A,G,L,S,Ste) 1.43E-06 (L,R)Mg24* 9.04E-05 (S,L) 8.31E-05 (L)Mg25* 7.95E-05 (L) 7.44E-05 (L)Mn55* 5.25E-06 (L,Ste) 9.22E-06 (L)Cu63* 6.05E-07 (D,L) 2.28E-06 (Can,L)Cu65* 1.41E-06 (A,D,L) 3.71E-06 (Can,G,L,R,S)Co59* 1.36E-06 (Ste) 1.45E-06Sr86* 1.10E-02 (B,Car,E) 1.45E-02 (B,Car,E)

Isotope Denoever Oria

Ba138* 5.49E-06 (B,Car,L,R,S) 5.76E-06 (L)Zn68 7.07E-06 6.11E-06Zn66* 8.49E-06 4.63E-06 (L)Rb85* 2.12E-05 (Can) 1.68E-05 (Can,G,S)Li7* 1.40E-06 (L) 1.65E-06 (L)Mg24* 7.47E-05 (L) 7.57E-05 (L,S)Mg25* 7.33E-05 (L) 6.97E-05 (L)Mn55* 6.57E-06 (L) 6.44E-06 (L)Cu63* 2.63E-06 (Can,Car,G,L,R,S,Ste) 6.12E-07 (L)Cu65* 4.31E-06 (Can,G,R,S,Ste) 1.51E-06 (L)Co59* 1.32E-06 1.77E-06Sr86* 1.45E-02 (B,Car,E) 1.28E-02 (B,Car,E,G)

Isotope SteMaries Burrishoole

Ba138* 4.25E-06 (L) 2.99E-06 (A,D,G,L)Zn68 7.41E-06 3.56E-06Zn66* 3.12E-06 (Can,Car,L) 2.99E-06Rb85* 3.51E-06 (G,S) 3.77E-05Li7* 1.60E-06 (Can,L,R) 1.00E-06 (L,S)Mg24* 1.05E-04 (L) 1.10E-04 (L)Mg25* 9.66E-05 (L) 7.41E-05 (L)Mn55* 8.07E-06 (L,R) 7.89E-06 (L)


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(Table 6). Factor 2 had high loadings from Cu63 andMg24 but did not contribute significantly to theseparation among samples (glass eels ⁄ elvers groupand leptocephali).

Using a quadratic discriminant function, leptoceph-ali samples were accurately classified as leptocephaliin 81% of the cases (Table 7). However, glass eel andelvers classification rates per each site were relativelylow, with a mean jackknifed accuracy estimate of 42%for the eleven sites gathered. No trend was found formisclassified samples to be classified to the nearestalternative sample site.

Discussion

Concentrations of individual elements in the otoliths ofglass eels and elvers did not vary among three annualcohorts collected in eleven European locations, sug-gesting that these natural markers were relatively stable

over several spawning periods. The latter result wasfurther confirmed by the inability of the quadraticdiscriminant analysis to discriminate among spatial andtemporal samples of A. anguilla glass eels and elvers.More importantly, misclassified individuals were notnecessarily identified as being from adjacent continentalrivers. Hence, we conclude that those specimens ofA. anguilla glass eels ⁄ elvers collected during threenonconsecutive years were born on a single spawningsite and that they, as leptocephali, migrated randomlytoward the continental distribution range of the species.This hypothesis is fully consistent with the most recentgenetic studies showing no evidence for a geographicalsubdivision in A. anguilla population (Dannewitz et al.2005; Maes et al. 2006; Pujolar et al. 2009).

Leptocephali were sampled during a short timeperiod (April 2007) and in a restricted area. Their sizesranged from 12 to 15 mm, which approximatelycorresponds to 20–30 days after hatching (Kuroki et al.

Table 5. (Continued).Isotope SteMaries Burrishoole

Cu63* 9.74E-07 (D,L) 1.57E-06 (L)Cu65* 2.21E-06 (D,L) 5.64E-06Co59* 1.64E-06 (B,Can,Car,E,G,R,S) 9.37E-07 (Ste)Sr86* 1.23E-02 (B,Can,Car,E,G,S) 1.19E-02 (A,Can,D,G,O,R,S,Ste)

Isotope Erne Carlingford

Ba138* 3.43E-06 (L) 3.53E-06 (D,L)Zn68 3.23E-06 4.59E-06Zn66* 2.59E-06 4.00E-06 (A,Ste)Rb85* 4.41E-05 (Can) 3.88E-05 (Can)Li7* 1.37E-06 (L) 1.24E-06 (L,S)Mg24* 9.58E-05 (L,S) 1.33E-04 (L)Mg25* 7.31E-05 (L) 1.16E-04 (L)Mn55* 7.16E-06 (L) 8.86E-06 (L)Cu63* 1.00E-06 (L) 9.91E-07 (D,L)Cu65* 2.30E-06 3.27E-06 (L)Co59* 9.03E-07 (Ste) 1.23E-06 (Ste)Sr86* 1.18E-02 (A,Can,D,G,L,O,R,S,Ste) 1.15E-02 (A,Can,D,G,L,O,R,S,Ste)

Fig. 4. Plot of the first two canonical dis-criminant functions summarising variationsin trace element signatures of Anguillaanguilla glass eels ⁄ elvers and leptocephaliotoliths. Glass eels and elvers are repre-sented by their capture locations (seeTable 1) and leptocephali are stated as‘lepto.’ in the legend.

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2006) and only relatively short migration distances(Bonhommeau et al. 2009c; Kettle & Haines 2006).Considering the relatively low spatio-temporal vari-ability of chemical concentrations in the central gyre ofthe Sargasso Sea surface waters (Bruland & Franks1983; Jakuba et al. 2008; Shiller 1997), we expected tofind a homogenous chemical fingerprint among lepto-cephali, and a similar signature for glass eels in thefollowing period with a time lag of 1 year, according toageing based on otolith microstructure (Lecomte-Fin-iger & Yahyaoui 1989) or 2–3 years according totransport models (Bonhommeau et al. 2009a; Kettle &Haines 2006). These characteristic chemical finger-prints were not however found in the subsequent 2008and 2009 glass eel ⁄ elver samples. Most of the elementratios in leptocephali otoliths were higher than in glasseels and highly variable among leptocephali.

Such a variance among leptocephali chemicalfingerprints could be explained by water chemistryof the specific western STCZ of the Sargasso Sea orparticular environmental parameters such as tempera-ture and salinity (Elsdon & Gillanders 2002; Fowleret al. 1995). Hence, experiments to test the response ofotolith trace element composition to variations in

water composition and temperature are ideally re-quired. Diet (zooplankton and ⁄or phytoplankton)might also play a role in otolith element compositionas leptocephali show a unique pattern of developmentand metabolism (Pfeiler 1999). More work is obvi-ously needed to identify the mechanisms that regulatethe differences in trace element signatures amongleptocephali.

However in this study, we used empirically derivedsignatures as natural tags of spawning locations withthe assumption that the elemental microchemistry atthe feeding check in otoliths of glass eels and elversis mainly influenced by the environmental chemistryof the spawning site(s). The otolith feeding checkregion analysed was the same for leptocephali andglass eels ⁄ elvers, thus the discrepancy between theirotolith fingerprints would be expected to reflectdifferences in the chemical composition of their natalenvironments and therefore suggested that severalspawning areas with different water composition mayexist. Currently it is widely accepted that spawningonly occurs in the Sargasso Sea, however, differentplaces within the Sargasso Sea can qualify forbreeding sites and there may be some spawningareas that increase the overall probability of successin migrating across the Atlantic Ocean (Bonhommeauet al. 2009a,c; Kettle & Haines 2006). Recruitmentmigration success was also found to depend onspawning location for Japanese eel (Kimura &Tsukamoto 2006). In the case of the European eel,the models proposed by Kettle & Haines (2006) andBonhommeau et al. (2009a), suggested that thesuccess of larvae in crossing the Atlantic Oceanand reaching particular regions of Europe is enhancedfor leptocephali starting from more westerly locationsin the Sargasso Sea than our sampling area. This ismainly due to the proximity of the Gulf Streamwhich permitted larvae to enter the Gulf Stream inless time than those more easterly spawned that drift

Table 6. Canonical coefficients for canonical variates (CV) 1 and 2 for 11isotopes examined in otolith core region among all samples (glasseels, elvers and leptocephali).

Element CV1 CV2

Mn55 0.2132 )0.1522Sr86 0.1027 )0.5508Ba138 )0.2258 )0.0896Zn66 )0.0221 )0.2108Rb85 0.0004 )0.1129Co59 )0.1219 0.1941Li7 0.4172 0.2319Mg24 0.5555 1.2016Mg25 0.2143 )1.0286Cu63 )0.0992 0.4037Cu65 )0.0210 )0.3452

Table 7. Results of the quadratic discriminant function analysis for classifying glass eels ⁄ elvers and leptocephali to the location of Anguilla anguilla samplestaken across Europe and Sargasso Sea using 24Mg, 25Mg, 63Cu, 65Cu, 59Co, 86Sr, 55Mn, 66Zn, 85Rb, 7Li and 138Ba as dependant variables. Glass eels and elvers arerepresented by their capture locations (see Table 1) and leptocephali are stated as ‘lepto.’ in the legend. Jack-knifed classification accuracies (in percent) andsamples sizes (in parentheses) are presented. The first column provides the capture location and the lines report the classification results.

% Lepto. Aiguillon Burrishoole Canet Carlingford Denoever Erne Gironde Oria Ringhals Severn SteMarie

Lepto. (16) 81 0 0 0 0 0 0 0 0 0 0 0Aiguillon (22) 0 18 0 0 0 6 0 0 11 2 2 10Burrishoole (9) 0 0 33 0 7 0 0 0 0 0 0 0Canet (45) 0 9 11 40 4 17 11 13 21 13 2 10Carlingford (28) 6 0 33 11 68 6 33 4 0 4 0 0Denoever (19) 0 0 0 2 0 11 0 0 0 0 0 0Erne (9) 0 0 22 2 4 0 33 0 0 0 0 0Gironde (45) 0 5 0 2 7 11 11 51 5 7 27 0Oria (19) 0 18 0 0 0 11 0 0 16 0 0 5Ringhals (45) 0 27 0 29 4 22 0 20 32 69 7 5Severn (45) 13 9 0 11 7 6 11 9 5 4 60 5SteMaries (21) 0 14 0 2 0 11 0 2 11 0 2 67


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in the slow currents of the Sargasso Sea (Kleckner &Mccleave 1982). Indeed, larvae with a long migrationduration, experience high mortality relative to larvaewith shorter migration duration. For many marinefish species, the major source of mortality occursduring the larval stage (Sifa & Mathias 1987); hencewe hypothesised that the leptocephali sampled in thisstudy had a low probability of reaching Europe orNorth Africa, while the sampled glass eels weresuccessful migrants that were born in areas wherefavourable transport conditions occurred. In addition,recruitment failure may result from particularlyunfavourable environmental conditions and foodavailability in certain regions of the Sargasso Sea(Bonhommeau et al. 2008; Friedland et al. 2007),e.g., in the central and easterly area sampled duringthe Galathea 3 cruise.

Although the exact location of the spawning site(s)remains unknown, the results of this study provideencouraging indications that elemental fingerprints ofthe otolith core region have potential for discriminat-ing among A. anguilla spawning areas. Consideringthese facts, it seems to be reasonable at the moment, tosuppose that several spawning areas for the Europeaneel may exist. As long as the question has not beenanswered why the Sargasso Sea is so unique forAtlantic eels’ reproduction, and as long as the exactlocation has not been confirmed, the entire area couldbe seen as potential breeding sites.

Without additional studies combining genetic andotolith fingerprints and tracking migrating adults, wecan only speculate from the present study on thegeographic interpretations of European eel otolithmicrochemical signatures. However, the results sug-gest such studies may help clarify aspects of thespatio-temporal dynamics and relative levels ofsuccess of larval and silver eel migrations andspawning. These could be of benefit in informingmanagement and conservation of the European eel inthe light of the declines in recruitment seen in recentdecades.

As a future work, it would be of value to compareelemental signatures in the primordium of otoliths inemigrating silver eels from different sources withthose in leptocephali in the subsequent breedingseason in different areas in the Sargasso Sea andmeasure elemental signatures in increments from theprimordium outwards in larval, yellow and silver eelotoliths to examine spatio-temporal changes in chem-ical signatures.

Acknowledgements

Research was funded by the European project EELIAD (http://www.eeliad.com). Sampleswere kindly provided byD.Evans,R.Poole, A. Walker, H.Wickstrom, P.Sasal and the ‘UK Glass Eel

Ltd’. The first author was supported by Cemagref (EstuarineEcosystems and Diadromous Fish Research Unit) during thepreparation of the manuscript. Finally, we would like toacknowledge the input and efforts of the anonymous reviewerwho contributed greatly to improve the paper.

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An otolith microchemistry study of possible relationships between the origins of leptocephali of European eels in the Sargasso Sea and the continental destinations and relative migration