Molecular Ecology (2011) 20, 1333–1346

doi: 10.1111/j.1365-294X.2011.05011.x

FROM THE COVER

All roads lead to home: panmixia of European eel in the
Sargasso Sea
T H O M A S D . A L S , * 1 M I C H A E L M . H A N S E N , ‡1 G R E G O R Y E . M A E S , § M A R T I N C A S T O N G U A Y , –L A S S E R I E M A N N , * * 2 K I M A A R E S T R U P , * P E T E R M U N K , †† H E N R I K S P A R H O L T , § § R E I N H O L D
H A N E L – – and L O U I S B E R N A T C H E Z * * **National Institute of Aquatic Resources, Technical University of Denmark, Vejlsøvej 39, DK-8600 Silkeborg, Denmark,‡Department of Biological Sciences, Aarhus University, Ny Munkegade 114, DK-8000 Aarhus C, Denmark, §Laboratory of
Animal Diversity and Systematics, Katholieke Universiteit Leuven, B-3000, Leuven, Belgium, –Institut Maurice-Lamontagne,
Fisheries and Oceans Canada, PO Box 1000 Mont-Joli, QC G5H 3Z4, Canada, **Department of Natural Sciences, Linnaeus
University, SE-39182 Kalmar, Sweden, ††National Institute of Aquatic Resources, Technical University of Denmark, DK-2920
Charlottenlund, Denmark, §§International Council for Exploration of the Sea, DK-1553 Copenhagen, Denmark, ––Institute ofFisheries Ecology, Johann Heinrich von Thünen-Institut (vTI), Federal Research Institute for Rural Areas, Forestry and Fisheries,
Palmaille 9, 22767 Hamburg, Germany, ***Département de Biologie, Institut de Biologie Intégrative et des Systèmes (IBIS),
Pavillon Charles-Eugène-Marchand, 1030, Avenue de la Médecine, Université Laval, QC G1V 0A6, Canada

Abstract
European eels (Anguilla anguilla) spawn in the remote Sargasso Sea in partial sympatrywith American eels (Anguilla rostrata), and juveniles are transported more than 5000 km
back to the European and North African coasts. The two species have been regarded asclassic textbook examples of panmixia, each comprising a single, randomly mating
population. However, several recent studies based on continental samples have foundsubtle, but significant, genetic differentiation, interpreted as geographical or temporal
heterogeneity between samples. Moreover, European and American eels can hybridize,but hybrids have been observed almost exclusively in Iceland, suggesting hybridization
in a specific region of the Sargasso Sea and subsequent nonrandom dispersal of larvae.
Here, we report the first molecular population genetics study based on analysis of 21
microsatellite loci in larvae of both Atlantic eel species sampled directly in the spawningarea, supplemented by analysis of European glass eel samples. Despite a clear East–West
gradient in the overlapping distribution of the two species in the Sargasso Sea, we only
observed a single putative hybrid, providing evidence against the hypothesis of a wide
marine hybrid zone. Analyses of genetic differentiation, isolation by distance, isolation
by time and assignment tests provided strong evidence for panmixia in both the Sargasso
Sea and across all continental samples of European eel after accounting for the presenceof sibs among newly hatched larvae. European eel has declined catastrophically, and our
findings call for management of the species as a single unit, necessitating coordinatedinternational conservation efforts.
Keywords: Anguilla, hybridization, individual assignment, isolation by distance, panmixia,
spawning migration
Received 6 October 2010; revision received 12 December 2010; accepted 14 December 2010

Introduction
Correspondence: Michael M. Hansen, Fax: +45 89422722;
E-mail: michael.m.hansen@biology.au.dk
1
These authors contributed equally to this work.
2
Present address: Section for Marine Biology, University of
Copenhagen, Strandpromenaden 5, DK-3000 Helsingør,
Denmark.
 2011 Blackwell Publishing Ltd

Different species can exhibit widely different levels ofgenetic differentiation over a given spatial scale, depending on the vagility and life history of the organism andthe environment it inhabits (Hamrick & Godt 1996;
Waples 1998). However, even highly vagile organisms

1334 T . D . A L S E T A L .
such as many marine organisms persisting in environments without obvious barriers to gene flow typicallyexhibit low, but statistically significant genetic differentiation (Nielsen et al. 2009a). Testing correlation
between environmental factors and genetic structure(landscape genetics) and analysing footprints of selection at the genomic level (population genomics) havefurthermore led to a growing understanding that oceanographic features may shape the structure of populations of marine high-gene flow species (Jørgensen et al.
2005; Fontaine et al. 2007; Selkoe et al. 2010) and that
local adaptation may occur (Nielsen et al. 2009b).
Hence, it would be unexpected to find species that arewidespread, are distributed across different environments and yet are truly panmictic.
The spawning biology and population genetic structure of the European eel (Anguilla anguilla) is a classicalscientific mystery. Adult eels are distributed in freshwater and coastal regions, ranging from Subarctic environments in the Kola Peninsula and North Cape innorthern Europe to subtropical environments in Morocco and the Mediterranean regions of Egypt (Tesch2003). Yet, for centuries, the spawning places of European eel remained unknown. It was not until the early
20th century that Schmidt (1923) identified the southern
Sargasso Sea as the spawning area of both Europeanand American eels (A. rostrata). Since that time, spawning has been demonstrated to take place in frontal
zones generated by the Subtropical Convergence,extending more than 1000 km from East to West (Kleckner & McCleave 1987) (Fig. 1). The larvae are subsequently transported by the Gulf Stream and otheroceanic currents to the coastal and freshwater foraging

areas in Europe ⁄ North Africa and North America,
respectively, with European eel migrating a much
longer distance (>5000 km) than American eel(2000 km) (Tesch 2003).
The issue of panmixia vs. population genetic differentiation in European eel is highly controversial. Schmidt(1923) was able to separate European and Americaneels based on vertebrae or myomere counts but
observed no geographical differentiation within species.
Later studies using allozyme and mitochondrial DNA
markers did not allow the rejection of the panmixia
hypothesis in either species (Avise et al. 1986; Lintas
et al. 1998; van Ginneken & Maes 2005). More recently,
however, microsatellite marker studies of European eel(Daemen et al. 2001; Wirth & Bernatchez 2001) reported
low, but significant, genetic differentiation and weak,
but significant, isolation by distance, suggesting geographical genetic differentiation and providing evidence
against panmixia. This conclusion was later questioned
by studies that found differentiation between temporal
samples of glass eels (metamorphosed juvenile eels)
arriving at different times at the same locales, but not
on a geographical scale (Dannewitz et al. 2005; Maes
et al. 2006) or found slight temporal variation between
cohorts of adult eels but no geographical differentiation(Palm et al. 2009). These observations suggest genetic
differentiation caused by spawning time differences
and a strong variance in reproductive success amongindividuals and spawning groups in the Sargasso Sea,
leading to genetic differentiation between different ‘arrival waves’ of glass eels.
Resolving the panmixia controversy has become evenmore important because of recent drastic declines of the

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StreamFig. 1 Map showing the sampling stations in the Sargasso Sea. Stations
marked by black dots indicate that
European and ⁄ or American eel larvae
were sampled, whereas white dots indicate that no eel larvae were sampled.
The blue area outlines the approximate
spawning area (Schmidt 1923; Tesch2003), and the grey line indicates the
Subtropical Convergence zone.

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P A N M I X I A O F E U R O P E A N E E L I N T H E S A R G A S S O S E A 1335
species. Recruitment has declined to <5% comparedwith pre-1980 levels, primarily because of over-exploitation and habitat destruction (van Ginneken & Maes
2005; Astrom & Dekker 2007). European eel is now
listed as ‘critically endangered’ in the International
Union for Conservation of Nature (IUCN) Red List of
Threatened Species (http://www.redlist.org). However,it remains uncertain whether the species should bemanaged as one single panmictic unit or as several
demographically independent populations.
Another unresolved issue concerns hybridization.
European and American eels are known to hybridize,but hybrids are observed almost exclusively in Iceland(Avise et al. 1990; Albert et al. 2006). This observation
could indicate the presence of a distinct hybrid zone in
the Sargasso Sea, followed by nonrandom distributionof juvenile eels to coastal regions with hybrids predominantly being advected towards Iceland (van Ginneken& Maes 2005). Alternatively, if the two species exhibit
different duration of the larval phase, then hybrids
could exhibit an intermediate length larval phase, hence
undergoing metamorphosis to glass eels in the ocean
close to Iceland, situated between continental North
America and Europe (Avise et al. 1990; Albert et al.
2006).
Investigation of both panmixia and hybridization has
so far been based exclusively on continental samples ofjuvenile or adult eels. However, the reproducing unit is
the appropriate unit for making inferences regarding
the population structure of a species (Bowen et al. 2005;
Carlsson et al. 2007). These issues prompt direct andthorough reassessments of the population structure andhybridization at the breeding location. Thus, we conducted a study based on samples taken directly in the
spawning area in the Sargasso Sea, which are supplemented with samples of European glass eels from theirmajor distributional range (from Iceland to Morocco).

Materials and methods
Sampling and molecular analyses
Eel larvae were sampled in the Sargasso Sea in March–
April 2007 during the Danish Galathea 3 marine expedition. Sampling was conducted at 37 stations distributed
across four separate transects, along the longitudes
64W, 65W, 67W and 70W (Fig. 1 and Fig. S1, Supporting information). For detailed sampling procedures,
we refer to the study by Munk et al. (2010). In total, thesamples covered 600 km of the Subtropical Convergence frontal zone in the Sargasso Sea, extending from
East to West (Fig. 1). Larvae were sorted, measured to
the nearest millimetre and stored in RNALater (Qiagen, Hilden, Germany) or 96% ethanol.
 2011 Blackwell Publishing Ltd

DNA was extracted (AllPrep DNA ⁄ RNA kit, Qiagen
or E.Z.N.A. kit; Omega Biotek, Norcross, GA, USA)
from the entire larva for molecular species identification
and analysis of microsatellite markers. European eel,
American eel and several species of non-anguillid eels
spawn in the Sargasso Sea (Castonguay & McCleave1987). Individual larvae were identified to species, i.e.
European and American eels, based on analyses of themitochondrial cytochrome b gene (Trautner 2006), thenuclear 5S rRNA gene (Pichiri et al. 2006), and genotype
data for 21 microsatellite markers, applying Bayesianmodel-based clustering as implemented in STRUCTURE
2.3.1 (Pritchard et al. 2000). A total of 480 potential
Anguilla larvae were sampled, among which 92 individuals either did not amplify for the three different typesof markers or produced band or allelic patterns completely different from those expected for European andAmerican eels. On closer inspection, these individuals
typically exhibited morphological features (e.g. pigmentation) providing further evidence that they were larvaeof non-anguillid eels. In total, 271 European and 117
American eel larvae were identified.
A total of 1010 European glass eels were sampled at
fourteen geographical locations ranging from Iceland in
the north to Morocco in the south. Some of these samples have also been analysed in other studies (Wirth &Bernatchez 2001; Maes et al. 2006), but based on different sets of microsatellite markers. From four of the locations, temporal samples were available (see Fig. 2 forsampling locations, sample sizes and sampling years).
DNA was extracted using the E.Z.N.A. kit (Omega
Biotek).
The samples of eel larvae and glass eels were genotyped using 21 microsatellite DNA markers: Ajms03,
Ajms06 and Ajms10 (Tseng et al. 2001); Ajtr27, Ajtr37,
Ajtr42 and Ajtr45 (Ishikawa et al. 2001); Ang101, Ang114
and Aro63 (Wirth & Bernatchez 2001); Aan01, Aan03
and Aan05 (Daemen et al. 1997); AangCA58, AangCT53,
AangCT68, AangCT76, AangCT82, AangCT87, AangCT89(Wielgoss et al. 2008); AjFABP, which is currently
unpublished. The primers AjFABP-F: GGCAGTCGATGGAAACAAAC, AjFABP-R: CGTGTGCTCACAACGTTACC were developed based on Anguilla japonica
sequences of the heart fatty acid binding protein(H-FABP) gene (GenBank accession number AB039666.3).
Markers were amplified in five multiplexes containing
3–8 loci using the Qiagen PCR multiplexing kit according to the manufacturer’s recommendations. We used
0.5 lL of DNA template in 13 lL reactions, an annealing
temperature of 57 C and 30 PCR cycles. Amplified fragments (0.5–0.8 lL) were added to 10 lL of Hi-Di Formamide and LIZ size standard solution (100:1) andanalysed using an automated sequencer (ABI 3130
Genetic Analyser; Applied Biosystems, Foster City, CA,

1336 T . D . A L S E T A L .
Fig. 2 Map showing the sampling locations, sampling years and sample sizesof European glass eels.

Vogslækur
02-07-2001 (n = 55)

Ringshals
03-04-2001 (50)

Den Oever
25-05-2001 (51)Burrishoole
01-02-2001 (55)
11-02-2005 (30)14-03-2005 (30)Yzer

08-04-2001 (51)Loire 02-04-2001 (51)Adour05-12-2001 (60)24-02-2002 (59)

Rio Lima
21-07-2001 (51)

Oued Sebou
28-04-2001 (51)24-02-2003 (59)04-03-2003 (60)14-04-2003 (59)Tour Du Valat
15-01-2001 (53)

Tiber
Canet

12-01-2008 (40)23-01-2008 (10)1999 (49)

Salses-Leucate

1999 (41)

Moulonya1999 (45)USA). Genotypes were scored using GeneMapper version 4.0 (Applied Biosystems).

Analysis of genetic variation
For each microsatellite locus and within each sample,
the expected and observed heterozygosity and numberof alleles were estimated using Arlequin version 3.11
(Excoffier et al. 2005). Deviations from Hardy–Weinberg
equilibrium because of heterozygote deficits were tested
by permutation tests to determine if FIS > 0, as implemented in FSTAT 2.9.3.2 (Goudet 1995).

taining all 388 eel larvae and all 1010 European glass
eels. An admixture model was applied without using
prior information on population structure and assumingthat allele frequencies are independent. The number ofclusters (k) with the highest posterior probability was
identified using replicate runs assuming k from 1 to 10.
A burn-in length of 100 000 steps was followed by
500 000 steps to obtain accurate parameter estimates,and 10 replicate analyses were conducted for each valueof k to verify the consistency of the results. The most
likely number of clusters was evaluated based on theposterior probability of k and the Dk method (Evanno
et al. 2005).

Model-based clustering
Bayesian model-based clustering of multi-locus genotype data, as implemented in STRUCTURE 2.3.1 (Pritchard et al. 2000), was used for assigning individuals toclusters corresponding to species and for identifying
potential hybrids. The analyses were conducted for two
data sets: (i) one consisting of 388 European and American eel larvae from the spawning area and (ii) one con-

Analysis of genetic population structure
The subsequent analyses were conducted for European
eel only because of limited geographical sampling ofAmerican eel (see Fig. 1 and Fig. S1, Supporting information).
The statistical power to detect genetic heterogeneity
at various true levels of differentiation was evaluated 2011 Blackwell Publishing Ltd

P A N M I X I A O F E U R O P E A N E E L I N T H E S A R G A S S O S E A 1337
for the present set of samples, number of loci and allele
frequencies using POWSIM 4.0 (Ryman & Palm 2006).
POWSIM simulates sampling of genes from a specified
number of populations that have diverged because of
random drift into an expected predefined level of differentiation (measured as FST). Samples were drawn fromthe simulated populations and used for testing genetic
homogeneity using Fisher’s exact test based on all locisimultaneously. Estimates of power were obtained as
the proportion of significant outcomes when repeating
the simulations 1000 times for each level of FST. POWSIM cannot handle markers with more than 50 alleles.
Therefore, rare alleles were pooled for the few markers
that exhibited more than 50 alleles (Table S1, Supporting information).
Global FST (Weir & Cockerham 1984) was computed
and genetic differentiation among samples tested using
the G-test (Goudet et al. 1996) as implemented in the
ADEGENET 1.2-3 package (R) (Jombart 2008). Pairwise
FST for different combinations of samples was estimatedusing the Fstat function of the GENELAND-package (R)(Guillot et al. 2005), which computes F-statistics using
Weir & Cockerham’s (1984) estimators. Estimation of
two- and three-level hierarchical F-statistics was performed using the HIERFSTAT 0.04-4 (Goudet 2005)
package in R. The significance of F-statistics was tested
by permuting individuals 1000 times among samples
and, for the hierarchical analyses, by permuting individuals among samples and samples among groups.
Isolation by distance was tested by pooling samples
within transects, calculating pairwise values of
FST ⁄ (1)FST) (Rousset 1997) and measuring East–West
geographical distance between transects. The significance of the correlation coefficients between genetic
and geographical distance was tested by permuting
individuals among samples and estimating pairwise FST
and r2 20 000–50 000 times in R, and comparing
observed values with the obtained null distribution ofr2 values.
Isolation by distance could be masked by different
ages of larvae, where older larvae would be expected
to have drifted longer from East to West along thethermal fronts (Fig. 1), whereas younger larvae would
be sampled closer to where they were born. It is also
possible that temporal reproductive isolation (isolation
by time; Hendry & Day 2005) could be misidentified
as isolation by distance, if different spawning events
had occurred at random along the thermal fronts. We
therefore tested for isolation by distance controllingfor possible isolation by time, and for isolation bytime controlling for possible isolation by distance. Specifically, we tested for correlations between pairwise
individual genetic distances, geographical distances(latitudinal distance, longitudinal distance or total dis 2011 Blackwell Publishing Ltd

tance) and temporal ‘distances’ (i.e. differences ofhatch date between individuals) using the partial
Mantel test (Legendre & Legendre 1998) of the NCF
package in R. P-values for the partial Mantel tests
were based on 1000 permutations. Rousset’s (2000)
genetic distance between individuals was estimatedusing SPAGeDi 1.3 (Hardy & Vekemans 2002). Hatch
date was calculated from capture date and length
measurements (see Fig. S2, Supporting information) byassuming a hatch size of 3 mm and growth rate of
0.6 mm ⁄ day until a length of 8 mm, followed by agrowth rate of 0.38 mm ⁄ day (personal communication:
Jonna Tomkiewicz, National Institute of Aquatic
Resources, Technical University of Denmark; Castonguay 1987; Kuroki et al. 2006). Some eel larvae were
measured immediately after sorting and subsequently
stored in RNAlater, whereas others were recoveredfrom plankton samples stored in 96% ethanol for
approximately 6 months before length measurements.
Assuming that eel larvae shrink to approximately 85%of their original length when stored in ethanol (Peter
Munk, National Institute of Aquatic Resources, Technical University of Denmark, personal observation), we
increased the length measurements of these samples
accordingly. Assuming different rates of shrinking(e.g. 90%) did not provide qualitatively different
results.
To rule out the effect of sampling families, half-sibs
and full-sibs within samples of eel larvae were identified by applying the maximum-likelihood method
implemented in COLONY 2.0.0.1 (Wang 2004; Jones &Wang 2010). Pairs of individuals identified as sibs but
sampled at different stations were considered unlikely
to be real sibs, whereas individuals identified as sibs
and sampled in the same haul were considered possibletrue sibs. The analysis was based on the full likelihood
method and the ‘long length of run’ and ‘high likelihood precision’ options implemented in COLONY. Relevant statistical analyses (FST, isolation by distance andpartial Mantel tests) were repeated by excluding one
randomly chosen individual from each identified pairof half- or full-sibs, thereby investigating the effect ofsiblings.
Glass eels were sampled from the major distributional range of adult European eel, and all putative
spawning populations of the European eel were probably represented in this sample. However, the possibility
that the samples from the spawning area do not represent the entire eel population is a potential risk. To
exclude the possibility of the presence of such unsampled ‘ghost populations’, we pooled all European eel
larval samples from the Sargasso Sea and estimated theprobability of assignment to this pooled sample foreach individual glass eel. This analysis was conducted1338 T . D . A L S E T A L .
Transect 1
Admixture proportions (q)Transect 2
Admixture proportions (q)Transect 3
Admixture proportions (q)Transect 4
Admixture proportions (q)0.0 0.2 0.4 0.6 0.8 1.0

0.0 0.2 0.4 0.6 0.8 1.0

0.0 0.2 0.4 0.6 0.8 1.0

0.0 0.2 0.4 0.6 0.8 1.0

Fig. 3 Individual admixture proportions and their 90% confidence intervals for Sargasso Sea eel larvae sampled at each transect,estimated using STRUCTURE (Pritchard et al. 2000) and assuming the presence of two clusters corresponding to European andAmerican eels (q denotes the probability of belonging to the European eel cluster). Values for American eel are presented in blue, forEuropean eel are shown in red, and a single putative hybrid is indicated by orange.

using the simulation test by Paetkau et al. (2004) basedon 100 000 simulated individuals, as implemented in
GENECLASS 2 (Piry et al. 2004).

Results
Distribution of European and American eel larvae
The geographical distribution of the sampled 271 European and 117 American eel larvae showed a predominance of American eel in the West, gradually replacedby European eel towards the East (Fig. S1, Supporting

information and Fig. 3). The North–South distributionof individuals across transects clearly showed a concentration of larvae in the main frontal zone, confirmingthat larval recruitment is associated with thermal fronts(Kleckner & McCleave 1987) (Fig. S1, Supporting information). Therefore, genetic differentiation would be
expected to occur along an East–West axis and wepooled samples into four transect samples. These fourtransect samples formed the basis of all subsequent
analyses based on Sargasso Sea eel larvae. However,
the alternative grouping along the North–South axis
was also examined.
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P A N M I X I A O F E U R O P E A N E E L I N T H E S A R G A S S O S E A 1339

Analysis of genetic variation
Number of alleles, observed and expected heterozygosity for each of the 21 microsatellite loci in each sample
are shown in Table S1, Supporting information. Tests
for Hardy–Weinberg equilibrium based on FIS estimatesyielded 11 significant outcomes in European eel andfour significant outcomes in American eel among 608
tests after false discovery rate correction (Benjamini &Yekutieli 2001) (Table S2, Supporting information).
Except for four significant outcomes among 25 tests
involving Ajms10 in European eel and two significant
outcomes among four tests involving AangCA58 in
American eel, there were no general tendencies towards
heterozygote deficits at the loci.

Model-based clustering and hybridization
Based on the analysis of microsatellite markers, significant genetic differentiation was found between European and American eel larvae (FST = 0.09, P < 0.001),
suggesting sufficient statistical power for detecting
hybrids. STRUCTURE (Pritchard et al. 2000) analysesof (i) the Sargasso Sea eel larvae and (ii) the combined data set of eel larvae and European glass eelsin both cases identified k = 2 as the most likely number of clusters, corresponding to the two species (datanot shown). Subsequent analysis of the European eel
samples suggested k = 1, thus providing no evidence
for intraspecific genetic differentiation (data notshown).
Obtained q-values (individual admixture proportions)with corresponding 90% confidence intervals (CI) were
inspected, and individuals with q-values close to 0 andlower 90% confidence limits overlapping with 0 wereidentified as American eel, whereas individuals with
q-values close to 1 and upper 90% CI encompassing 1.0
were identified as European eel. Admixed individuals(i.e. with intermediate q-values) for which the 90% CI
contained neither 0.0 nor 1.0 were considered to be
hybrids between the two species. Individual admixture
proportions for the Sargasso Sea eel larvae showed thatthe two species could be clearly separated (Fig. 3). Onlya single individual showed intermediate admixture proportions (q = 0.338, CI90% = 0.070–0.594), suggestingthat this was a hybrid. Among the 1010 European glass
eels, three putative hybrids were identified, including
one individual from Iceland (Fig. 4).

Genetic differentiation among European eel larvae
Larger and thus older eel larvae may have been born aconsiderable distance from where they were sampled.
Therefore, we analysed genetic differentiation using 2011 Blackwell Publishing Ltd

only individuals measuring <15 mm (n = 220; see
Fig. S2, Supporting information). An analysis of statistical power using POWSIM (Ryman & Palm 2006)
showed that the sample sizes and specific genetic markers applied were adequate for detecting a low level ofgenetic differentiation (FST = 0.0011) with a high probability (>0.9) (Fig. 5).
Very low and nonsignificant genetic differentiation
was observed among the four transect samples(FST = 0.00076, P = 0.0870, n = 220), and pairwise FST
values were also low and nonsignificant (Table S3, Supporting information). Significant correlation (r2 = 0.8175,
P = 0.0098) between genetic (FST ⁄ 1)FST) and geographical distance was nevertheless detected (Fig. 6a), potentially indicating East–West isolation by distance.
Sampling larvae only a few days old has a risk ofincluding sibs within the same hauls, which could create subtle, but significant, correlations. The method forreconstructing sibships implemented in COLONY(Jones & Wang 2010) identified two full-sib families at
two stations in the two westernmost transects, comprising three and two full-sibs, respectively (Fig. 7).
Twenty-two pairs of putative half-sibs were additionally identified within stations. It must be consideredhighly unlikely that individuals collected at different
stations are true sibs. Hence, comparing the number ofputative sibs within stations with the number of putative sibs sampled at different stations provides an indication of the accuracy of the COLONY analysis. No
full-sib pairs were identified involving individuals fromdifferent stations, and Fisher’s exact test comparing
observed and expected numbers of full-sib pairs yieldeda highly significant outcome (P = 0.00008). In contrast,
247 possible half-sib pairs were identified between stations, and the number of half-sib pairs within andbetween stations was not significantly different (Fisher’s
exact test, P = 0.6041). Hence, the identified full-sib
families are likely to represent real sibs, whereas severalof the putative half-sib pairs are likely to represent false
positives.
Random removal of all but one individual from eachpair of full-and half-sibs identified within stations substantially changed the results. Genetic differentiation
among the four transects decreased further (FST =)0.000287, P = 0.4825, n = 199), and the apparent isolation by distance became nonsignificant (r2 = 0.1804,
P = 0.4739; see Fig. 6a). As half-sibs are likely toinclude false positives, we further repeated the analysis
by only excluding one individual of each full-sib pair.
Again, this yielded low and nonsignificant genetic differentiation (FST = 0.00019, P = 0.4755, n = 217) andnonsignificant isolation by distance (r2 = 0.4728,
P = 0.1475). Finally, to further verify that the apparent
isolation by distance could be explained solely by1340 T . D . A L S E T A L .
1.0–log(p)

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Assignment probability(uncorrected p-values)

q-valueSignificant low probability

Putative hybrid

FDR critical P-value (1%) = 0.0013
1.0

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Tour-Du-Valat 15-01-2001

Tibre 1999

0.0

Fig. 4 Individual admixture proportions and assignment probabilities for individual glass eels from Europe and North Africa. Individual admixture proportions (q) with corresponding 90% confidence intervals, estimated using STRUCTURE (Pritchard et al. 2000),are shown in red for each individual. The analysis assumes k = 2 corresponding to European and American eels, and q denotes theprobability of belonging to the European eel cluster. Bars denote the probability of assigning an individual glass eel to the pooled
sample of European eel larvae from the Sargasso Sea, using a simulation test implemented in GENECLASS2 (Piry et al. 2004). The
green dotted line denotes the false discovery rate-corrected (Benjamini & Yekutieli 2001) critical P-value at the 1% significance level.
Note that the scale on the left y-axis represents )log10(P), and a high value corresponds to a low probability.

sampling of two full-sib families, we randomly
removed three unrelated individuals from the transect
samples where the sibs were identified, analysed isolation by distance and repeated this procedure 1000
times. The fitted isolation-by-distance line was very
similar to that obtained when full-sibs were included,
and the 95% confidence interval of simulated r2 values
exceeded the r2 value observed when full-sibs were
removed (Fig. 6b). Hence, we conclude that the apparent isolation by distance represents artefacts caused byinclusion of only a few sibs.
We further investigated if different groupings of samples, e.g. by pooling stations into four arbitrary groups
from north to south, or different thresholds for size oflarvae included in the analyses could affect the results.
In none of the cases, significant FST or isolation by distance was observed (Table S4, Supporting information).
Hierarchical analysis of genetic differentiation (Goudet2005) also yielded low and nonsignificant differentiation

among transects and among stations within transects(Table 1).
Some previous studies suggested that genetic differentiation in European eel reflects isolation by time
(Hendry & Day 2005) rather than distance (Dannewitz
et al. 2005; Maes et al. 2006). We therefore conductedpartial Mantel tests (Legendre & Legendre 1998) analysing the association between pairwise individual genetic
distance (Rousset 2000), geographical distance and ‘temporal distance’ (differences in estimated hatch date
between individuals). However, all associations werenonsignificant whether or not the effect of sibs withinsamples was removed, and whether or not longitudinal,
latitudinal or total geographical distances between individuals were considered (Table S5, Supporting information).
In total, the analysis of samples from the Sargasso
Sea yielded no evidence for genetic differentiation, isolation by distance or isolation by time.
 2011 Blackwell Publishing Ltd

(a)

0.000

0.6

175
r2 = 0.8
2 = 0.4728
rr2 = 0.1804−0.0040.4
0.2

Statistical power

0.8

0.004

1.0

P A N M I X I A O F E U R O P E A N E E L I N T H E S A R G A S S O S E A 1341

0.0

100

Genetic differentiation among glass eels
Nonsignificant genetic differentiation was found among
the total of 21 geographical and temporal samples ofglass eels (FST = 0.00024, P = 0.2464), and there was noevidence for isolation by distance (r2 = 0.0008,
P = 0.9283; see Fig. 6c). Constraining the analysis to theyear with most samples (2001) also showed nonsignificant genetic differentiation (FST = 0.00021, P = 0.5345)
and no evidence for isolation by distance (r2 = 0.0640,
P = 0.3612; see Fig. 6c). Three-level hierarchical F-statistics (Goudet 2005) provided no evidence for temporal
genetic differentiation between glass eels sampled indifferent years from the same site or between glass eels
sampled on different dates within the same year andsite (Table 1).
Hence, similar to the analyses of Sargasso Sea eel larvae, we found no evidence for genetic differentiation,
isolation by distance or isolation by time in continental
samples of European glass eels.

Relationships between Sargasso Sea eel larvae andcontinental glass eels
No significant genetic differentiation was observed
between the pooled sample of European eel larvae fromthe spawning area and glass eels from Europe andNorth Africa (FST = )0.00012, P = 0.2136, n = 1282). To
fully confirm that the samples of glass eels and eel larvae belonged to the same panmictic population, and to
exclude the possibility of unsampled ‘ghost populations’, the probability of assigning each individual glass 2011 Blackwell Publishing Ltd

600

(b)
2)

.53–0.9

0.000

I %: 0801 (C 95
r2 = 0.7
175
r2 = 0.88
r2 = 0.472−0.004

Fig. 5 Simulated statistical power for obtaining significant outcomes in tests of genetic differentiation involving four samples
and the specific marker characteristics and sample sizes of Sargasso Sea European eel larvae. Simulations were performed
using POWSIM 4.0 (Ryman & Palm 2006). The dotted line indicates the minimum level of genetic differentiation that can bedetected with 90% statistical power.

0.004

0.0020100

0.004

0.0015

FST

500

200

300
400
Pairwise distance (km)

500

600

(c)

0.002

0.00100r2 = 0.064

0.000

0.0005300
400
Pairwise distance (km)r2 = 0.0008−0.0020.0000

200

0

1000

2000
3000
4000
Pairwise distance (km)5000

Fig. 6 Analyses of isolation by distance based on correlating
genetic (FST ⁄ 1)FST) vs. geographical distance. (a) European eel
larvae <15 mm. Results including putative half- and full-sibs
are shown in blue (r2 = 0.8175, P = 0.0098), results obtained
when excluding one individual from each putative full- or
half-sib pair are shown in orange (r2 = 0.1804, P = 0.4739) andresults obtained when excluding one individual from each fullsib pair are shown in red (r2 = 0.4728, P = 0.1475). (b) The
mean isolation-by-distance line generated by randomly excluding three nonfull-sib individuals 1000 times (r2 = 0.7801,
CI95% = 0.53; 0.92) is shown in black. The shaded area represents 95% confidence limits of the black line. The isolation by
distance lines including putative half-and full-sibs (blue line)
and excluding one individual from each full-sib pair (red line)
have been added for comparison. (c) Continental samples ofglass eels. Results involving all samples are shown in blue
(r2 = 0.0008, P = 0.9283), and results encompassing only
samples from 2001 are shown in red (r2 = 0.0640, P = 0.3612).

1342 T . D . A L S E T A L .
T1 33 T2

16

30
29

17
19

28

T4

T3

A22

13
12

20
21
22

11

27
23
26
25

24
10

A21

Unrelated

9

A3

Halfsibs
Fullsibs

Table 1 Hierarchical F-statistics for eel larvae and glass eel
samples, as implemented in HIERFSTAT 0.04-4 (Goudet 2005).
All P-values are based on 1000 permutations
Data set
Larvae < 15 mm excl. sibs* (n = 188)
Among transects
FCT
Among stations within transect
FSC
Larvae < 15 mm excl. full-sibs* (n = 206)
Among transects
FCT
Among stations within transect
FSC
Glass eels all years† (n = 1010)
Among sites
FCT
Among years within sites
FSC
Among dates within years
FZS
Glass eels† (n = 463)
Among sites
FCT
Among years within sites
FSC
Among dates within years
FZS

F-valueP-value)0.00034
0.00223

0.218
0.506

)0.00011
0.00282

0.37720.3639)0.00024
0.00158)0.00112

0.428
0.072
0.349

)0.00022
0.00146)0.00099

0.456
0.110
0.156

*One individual from each half- or full-sib pair was excluded,
and stations with only one individual were excluded.
†
The first data set consists of all samples, whereas the second
only includes sites sampled at more than one time point:
Adour (France), Loire (France) and Burrishoole (Ireland);temporal samples from Canet were not considered because of
low sample size.

8

found in the occurrence of glass eels with significantly
low assignment probabilities and, in total, the presenceof unsampled populations not represented by the sample from the spawning area appears unlikely.
A20

Discussion
7

Fig. 7 Pairs of putative full-sibs (green) and half-sibs (red)identified within stations using COLONY 2.0.0.1 (Jones &Wang 2010). Black dots denote individual eel larvae, and individuals from the same stations are connected by lines.

eel to the pooled sample of European eel larvae fromthe spawning area was estimated. Only three individuals (0.3%) showed significantly low P-values (at the
1% level) after false discovery rate correction (Benjamini & Yekutieli 2001) (Fig. 4). Individual admixture
proportions estimated using STRUCTURE (Pritchard
et al. 2000) did not suggest that any of these individuals
were hybrids between European and American eels,
although three other putative hybrids were observed(Fig. 4). No geographical or temporal pattern was

The results of the study provide important information
pertaining to the interpretation of findings of European · American eel hybrids and for resolving the controversy of panmixia in European eel. We discuss theseissues in detail in the following section.

Hybridization between European and American eels
Our study documents that the spawning areas of thetwo species show considerable geographical overlap,
and ample opportunities for hybridization exist. Nonetheless, the finding of only a single putative hybrid
does not support the presence of a distinct marine
hybrid zone. The occurrence of hybrid eels in Iceland(Avise et al. 1990; Albert et al. 2006) is unlikely toreflect nonrandom advection of hybrid eel larvae froma specific region of the Sargasso Sea (van Ginneken &Maes 2005). However, it does not contradict the alternative hypothesis that larval phase duration is intermediate in hybrids when compared to the parental species(Avise et al. 1990; Albert et al. 2006). Although low infrequency (<1%), hybrids will be numerically important 2011 Blackwell Publishing Ltd

P A N M I X I A O F E U R O P E A N E E L I N T H E S A R G A S S O S E A 1343
because of the abundance and high fecundity of thespecies. This could lead to metamorphosis and settlement of substantial numbers of hybrid eel larvae halfway on the route towards Europe, corresponding to thegeographical location of Iceland.

Panmixia in European eel
This study represents the most comprehensive European eel data set to date and includes unique samplesof larvae from the spawning area, along with glass eel
samples from continental foraging areas. The results
offer new and decisive insights into the European eel
panmixia controversy (Daemen et al. 2001; Wirth & Bernatchez 2001; Dannewitz et al. 2005; Maes et al. 2006;
Palm et al. 2009). All results are in accordance with apanmixia scenario. Despite considerable statisticalpower, there was no evidence for genetic differentiation
among samples of larvae from the Sargasso Sea.
Although weak but significant isolation by distance wasobserved, it was demonstrated that this was an artefact
caused by inclusion of a few sibs in some of the samples. This illustrates the inherent problems when interpreting very low genetic differentiation as often
observed in marine organisms (Waples 1998); evensmall bias and artefacts may substantially affect results.
Also, there was no evidence for more subtle structurecaused by spawning time differences leading to isolation by time (Hendry & Day 2005).
The analysis of newly spawned eel larvae sampleddirectly in the spawning area represents an optimal
strategy for testing the panmixia hypothesis, provided
that the entire European eel population is represented in
the analysed samples. To ensure that no unsampled
populations exist, and to further test the panmixia
hypothesis, we genotyped the same microsatellite markers in glass eels sampled on various dates and from sites
ranging from Iceland to Morocco. Again, very low andnonsignificant genetic differentiation was observed andthere was no evidence for isolation by distance. These
results conflict with previous studies (Daemen et al.
2001; Wirth & Bernatchez 2001), potentially because of
temporal variation in the degree of isolation by distance
between years, and genetic diversity may have been lost
during the last 30 years of declining eel population size
(Astrom & Dekker 2007). However, three-level hierarchical analysis of genetic differentiation provided no evidence for temporal genetic differentiation between eels
sampled in different years from the same site or
between eels sampled on different dates within the same
year and site. Thus, in accordance with a recent study ofadult eels (Palm et al. 2009), we found no evidence for
geographical or temporal differences among samples ofglass eels in continental waters.
 2011 Blackwell Publishing Ltd

Finally, genetic differentiation between Sargasso Sea
eel larvae and continental glass eel samples was very
low and nonsignificant. Individual assignment of glass
eels to the total European eel larvae sample showedthat eels from the whole distributional range from
Northern Europe to North Africa could be accounted
for by spawning in the Sargasso Sea and that no unsampled ‘ghost’ populations were likely to exist.
The results suggest a random arrival of adult eels inthe spawning area and subsequent random distributionof larvae across the European and North African
coasts. This is in agreement with several biological features of the species. First, eel larvae are transported>5000 km by the Gulf Stream and other currents before
reaching Europe and North Africa (Tesch 2003). This
provides ample opportunities for mixing of aggregations of larvae from different parts of the spawning
region, although oceanographic modelling has suggested that retainment of larval aggregations may bepossible under some conditions (Kettle & Haines 2006).
Second, the early life of eel larvae is intimately linked
to the thermal fronts of the southern Sargasso Sea, andspawning must be assumed to take place in the frontal
zones (Kleckner & McCleave 1987; Munk et al. 2010).
The fronts are highly dynamic and undulate along an
East–West axis (Tesch 2003; Munk et al. 2010). Precise
homing of eels to their specific natal site in the Sargasso Sea therefore seems unlikely as this would conflict with the necessity of spawning in the thermal
fronts. Moreover, data on the spawning migration ofEuropean eel (Aarestrup et al. 2009) and on the recent
discovery of spawning individuals of the related species A. japonica and A. marmorata in the Pacific Ocean(Chow et al. 2009) suggest that both migration andspawning occur in the upper pelagic zone (<1000 m).
Hence, imprinting and homing to specific bottom
structures in the up to 5-km-deep Sargasso Sea appear
unlikely. In total, we propose that spawning Europeaneels exhibit a crude homing behaviour towards thefrontal zone region of the southern Sargasso Sea, but
fine-scale homing towards their precise natal sites does
not take place.
The finding of panmixia in such a widely distributed
species inhabiting environments ranging from Subarctic
to Subtropical climates must be considered highly unusual. In many organisms, genotype · environment interaction and selection over this geographical andenvironmental scale would be expected to lead to local
adaptation (Kawecki & Ebert 2004). However, local
selection occurring across the continental distributional
range within a generation would be obliterated in the‘genetic melting-pot’ of the Sargasso Sea. Indeed,within-generation local selection acting on genes in linkage disequilibrium with studied genetic markers could

1344 T . D . A L S E T A L .
potentially explain the weak clinal patterns interpreted
as genetic structure in previous studies (Daemen et al.
2001; Wirth & Bernatchez 2001). This issue could be further clarified by population genomics analyses (Luikart
et al. 2003) aimed at identifying genes under selection
in continental and Sargasso Sea samples.
Our findings have crucial implications for conservation of this endangered species. The recovery time ofthe species is expected to be very slow; ca. 80 years
even in the case of complete closure of fisheries(Astrom & Dekker 2007), stressing the need for imminent action. Panmixia within the species implies thatmanagement and conservation efforts must be coordinated at the transnational level, as over-exploitation in
one region will negatively affect the total European eel
population and decrease recruitment across the whole
distributional range.

Acknowledgements
We thank the Captains and crews of HMS Vædderen and the
Newfoundland Alert for assistance with sampling, Françoise
Daverat, Javier Lobón, Stefan Palm, Russell Poole, Alan
Walker, and Håkan Wickström for providing glass eels, KarenLise D Mensberg for help in the laboratory and Sebastién
Moore and two anonymous reviewers for constructive comments on the manuscript. The study was funded by the Villum
Kann Rasmussen Foundation, Elisabeth and Knud Petersens
Foundation, the European Union FP7 (EELIAD grant 212133),
the Danish Council for Independent Research| Natural Sciences (grant 09-072120), and the Danish Expedition Foundation. The present work was carried out as part of the Galathea
3 expedition under the auspices of the Danish Expedition
Foundation. This is contribution no. P72 of the Danish Galathea 3 expedition.

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The authors of this paper represent a multidisciplinary team ofscientists with research interests spanning from evolutionary
biology and population genomics to fish biology and biologicaloceanography. The research was undertaken as part of theDanish Galathea 3 marine expedition, which involved sampling of European and American eel larvae and oceanographical analyses in the Sargasso Sea during spring 2007. In
addition to analyzing the genetic population structure of European eel the research team has studied the spawning migration of adult eels using satellite data storage tags, the feeding1346 T . D . A L S E T A L .
biology of eel larvae using DNA barcoding of gut contents andthe importance of oceanic fronts in the Sargasso Sea for theearliest life stages of eels.

Data accessibility
Data deposited at Dryad: doi:10.5061/dryad.8155.

Supporting information
Additional supporting information may be found in the online
version of this article.
Table S1 Genetic variation at the 21 microsatellite markers foreach of the 25 samples of larvae and glass eels of European
and four samples of American eel larvae.

Table S3 Pair-wise FST between all pair-wise combinations ofsamples of European eel.
Table S4 Global FST values and corresponding correlation coefficients (r2) for genetic [FST ⁄ (1)FST)] vs. geographic distance.
Table S5 Partial Mantel tests of correlations between individual genetic distances and geographic and temporal distances(hatch date).
Fig. S1 Bar plots of the number of American and European eel
larvae sampled at each station in the Sargasso Sea.
Fig. S2 Length distributions for the analyzed American andEuropean eel.
Please note: Wiley-Blackwell are not responsible for the content
or functionality of any supporting information supplied by theauthors. Any queries (other than missing material) should bedirected to the corresponding author for the article.

Table S2 FIS estimates and tests for Hardy–Weinberg Equilibrium for each marker in each sample.

 2011 Blackwell Publishing Ltd