BACKGROUND
hybridization, the interbreeding of diagnosably divergent species, is a major focus in evolutionary studies  <cit>  as it is a key concept for understanding the demographic and evolutionary cohesiveness of natural populations  <cit> .
hybridization has great potential to rapidly introduce variability into a recipient population, if barriers to recombination can be overcome.
cooper  <cit>  put forth strong experimental evidence that sexual recombination speeds up the origin and spread of adaptations in an artificial environment compared to purely clonal strains of escherichia coli, thus supporting the theoretical predictions from the fisher-muller model  <cit> .
based on the evidence gathered from empirical data, including invasive species  <cit>  and signatures of massive horizontal gene transfer in a paradigmatic long-term asexual species  <cit> , it might be generalized that in order to quickly adapt, any mechanism of lateral gene transfer or recombination is highly favored in unstable or novel environments.
importantly, the same might apply for populations overlapping after a phase of allopatry, as is assumed to be the case for north atlantic eels  <cit> .
the two north atlantic eel species, anguilla anguilla  and a. rostrata  both have a remarkable catadromous life-cycle that comprises two long-range migrations through the open ocean, a continental growing phase, and a spawning stage in the sargasso sea  <cit> .
despite the key importance of the marine phase  <cit> , most of the scientific investigations focused on the continental phase, and only few data exist on the oceanic parts of the eel’s life-history.
to this day, migration routes and exact spawning sites of eels remain largely hypothetical  <cit> .
fifty years ago, to the great surprise of the scientific community, tucker  <cit>  suggested that the european eel should be considered an evolutionary dead-end of non-spawning individuals.
however, with the advent of new molecular markers the genealogical distinctness of the two species was largely clarified .
deligny and pantelouris  <cit>  and avise et al.
<cit>  argued for the continuation of the two-species-status, originally proposed by schmidt  <cit> .
this scenario was reinforced when mitochondrial genome sequences became available  <cit> .
recently, using miniaturized pop-up satellite archival transmitters , a small number of eels could be followed during their spawning migration off the european continental shelf towards the canary and azores current system.
the authors confirmed the eels’ daily vertical migrations between depths of 200–1000 m at a speed of five to 25 km per day  <cit> .
*reconsidered by koehn : listed markers agree with mendelian inheritance and have objectively interpretable banding patterns.
**statistically re-evaluated by applying χ2-statistics with correct degrees of freedom to test for hardy-weinberg-equilibrium.
atlantic eel stocks have rapidly declined by over 95% compared to the levels prior to  <dig>  <cit> .
as a consequence the european eel has been listed as critically endangered on the iucn red list  <cit> .
this dramatic decrease is attributable to a combination of factors, including habitat destruction, pollution, and over-fishing  <cit> , climate induced changes in the gulf stream circulation  <cit> , as well as the recent introduction and spread of anguillicola crassus, an exotic swimbladder nematode that was initially introduced along with infected japanese eels  <cit> .
disentangling the population structure of both north atlantic eel species thus has implications on international eel stock management both from conservation and fisheries perspectives.
advanced molecular tools permitted the collection of larger and more sensitive data sets at the population level .
however, despite huge sampling efforts these studies have come to different and even contradictory conclusions.
three independent studies reported isolation-by-distance  in a. anguilla which provided evidence against the long-held paradigm of panmixia for this species based on the measurement of very low, but nevertheless highly significant genetic structure within european eels  <cit> .
wirth and bernatchez  <cit>  suggested that ibd patterns are due to a stable temporal delay of spawning migration in eels from northern habitats, as distances are markedly extended compared to western and southern european populations.
alternatively, more than one spawning area might be used by different populations and, also, different currents might carry the leptocephali larvae back from the sargasso sea to their parent’s original freshwater habitats in europe.
thirdly, albeit seemingly less likely, assortative mating among regional groups might be responsible for the observed ibd patterns.
finally, maes et al.
<cit>  suggested that, given the random factors affecting spawning success in the open ocean, a sweepstake strategy  <cit>  might explain genetically patchy recruitment in sampling locations across europe  <cit> , leading to a weak but significant isolation-by-time  signal.
however, most recent studies did not find statistical support for population structure and rejected both ibd and ibt altogether, thus favoring panmixia in both atlantic eel species  <cit> .
in addition, temporal variation among recruits between different arrival waves within the same years significantly exceeded both the geographic and inter-annual genetic differentiation reported above  <cit> .
several points have been raised to explain the discrepancies between early evidence against panmixia and later studies.
palm et al.
<cit>  argued that genetic differentiation in the european eel might be largely explained by uncontrolled temporal variation between juvenile glass eel samples.
dannewitz et al.
<cit>  speculated that the use of different cohorts or life stages in the initial studies with strongest support against panmixia could have lead to artifactual isolation-by-distance patterns.
lastly, after evaluating the weak genetic differentiation among north atlantic eels using basic summary statistics, mank and avise  <cit>  concluded that the large overlap in their allelic frequencies is generated by extensive homoplasy associated with a mutation-driven saturation effect.
this argument casted doubts on the usefulness of rapidly evolving microsatellite loci for short term evolutionary and hybridization studies in eels.
while there exists strong empirical evidence for near panmixia in both species of north atlantic eels, not all questions could be successfully addressed.
first, it is difficult to see how saturated markers and patchy cohorts within sampling locations could generate statistically significant patterns of ibd over the whole distributional range  <cit> .
second, the same markers detected the highest signatures of admixture in icelandic samples  <cit>  are consistent with meristic and genetic data that suggested the existence of hybrids in northern eel habitats in general  <cit> .
this pattern suggests the existence of a hybrid zone in the atlantic that could lead to clines of admixture in both species of eel.
in fact, this hypothesis has recently been invigorated by a study that relied on an integrated demographic-genetic model and explicitly accounted for the different levels of larval and adult mixing during oceanic migrations  <cit> .
this model showed that even minimal levels of mixing among initially separate sub-populations during both larval dispersal or adult migration are sufficient to entirely erase any genetic differences among them.
building on this finding, the authors offer two explanations for why the geographic differentiation component might have been overestimated in the past:  a limited number of temporal recruits analysed,  introgression through inter-species hybridization and non-random dispersal.
there is a growing number of studies that show hybridization with molecular data  <cit> , but no study to date has systematically tested this in the north atlantic eels across their entire range of distribution.
traditionally, many zoologists are reluctant to consider hybridization as an important evolutionary process that generates new species, since the pre- and post-mating barriers to establishing f <dig> hybrids are often considerable  <cit>  .
however, there is a growing number of examples of reticulate evolution in nature  <cit> , particularly from plants and fungi .
a very well known feature of hybrid zones are coincidental changes at several independent characters, resulting in parallel frequency gradients , and such a linkage is measurably exemplified in north atlantic eel species, a. anguilla and a. rostrata.
here, cyto-nuclear disequilibrium has been identified in the narrow zone of species overlap in iceland  <cit> , which scales well with a transition zone based on vertebral counts, a nearly diagnostic trait between the two species of eels  <cit> .
more specifically, avise et al.
<cit>  estimated the hybrid fraction in iceland to be  <dig> to 4% .
later, albert et al.
<cit>  evaluated the extent of hybridization and tested for the occurrence of hybrids beyond the first generations, using  <dig> aflp markers.
a total hybrid fraction of  <dig> % was identified, of which 30% were considered to be later generation hybrids.
h
n
h, mitochondrial frequency of a. rostrata haplotypes in iceland; n, sampling size.
here, we investigate the extent of population genetic differentiation, and by performing simulations, ask whether ibd patterns might be produced by increasing inter-species hybridization and admixture clines.
such a scenario would also be able to explain the emergence of an ibt signal and fluctuating genetic structure signatures over time.
by combining and extending two available data sets  <cit>  genotyped at nine microsatellite markers, we investigated eels sampled from the whole north atlantic distribution area for signals of inter-species hybridization.
in a first step, we inferred the admixture proportions for each individual and tested if the most extreme ibd signal detected in european eels  <cit>  can be explained by latitudinal admixture and introgression clines using a simulation approach.
second, we quantified the amount of gene flow that would be necessary to generate the observed correlations, and, finally, we tested if incomplete lineage sorting or recent introgression are most likely to explain the observed lack of monophyly in most nuclear markers observed so far.
RESULTS
summary statistics
nine microsatellite loci were used to infer intra-specific relationships among north-atlantic eels.
inter-specific comparison of genetic differentiation for both fst and rst estimates were highly congruent with one another at  <dig>   and  <dig>  , respectively.
thus, microsatellite markers used appear to essentially evolve by addition or removal of simple repeats in a step-wise manner.
overall, levels of observed polymorphism were high in north atlantic eels, ranging from ho =  <dig>  at locus aro <dig> to ho =  <dig>  at locus ang <dig>  with a mean of ho =  <dig> .
the same was true for the average numbers of alleles when correcting for sample sizes, ranging from ar =  <dig>  in aro <dig>  to ar =  <dig>  at locus ang <dig>  none of the  <dig> pairwise tests for linkage disequilibrium among loci was significant after bonferroni correction  <cit> .
however, micro-checker <cit>  revealed the presence of null alleles for the two newly added loci ang <dig> and aro <dig>  this observation might explain the moderately high fis values for most sampling localities  and a systematic deviation from hwe for all populations.
however, after correction for null alleles using the algorithm freena <cit> , all but three out of  <dig> sampling locations were compatible with hwe after bonferroni correction  <cit>   and in each case where a significant deviation from hwe was detected, only a single locus contributed to the effect.
detection of hybrid eels in iceland
based on diagnostic restriction digests of cytb fragments,  <dig> out of  <dig> icelandic eels  carried american haplotypes , whereas none of the continental north atlantic eels showed restriction patterns corresponding to the other species .
thus, as already described, there appears to be a rather clear-cut sorting of mitochondrial lineages with the species boundaries  <cit> .
the divergence was less pronounced based on ancestry proportions at nuclear markers, which consistently separated american and european gene pools for the most likely number of populations, k =  <dig>  importantly, the average ancestry proportion of the  <dig> “suspects” in iceland  based on nine microsatellite markers was intermediate  compared to those generated from  <dig>  blind draws of  <dig> random individuals from either species , and thus these suspected mtdna hybrid individuals most likely represent true f <dig> hybrids.
in addition, the mean ancestry proportion of non-suspect icelandic eels was not significantly different from the european mean, albeit slightly shifted toward american eels .
when adding prior geographic information on continental stocks, no american expatriate  was detected in iceland , but two eels carrying a european mtdna haplotype were assigned as pure american given their nuclear data.
geographic admixture and introgression clines and their impact on ibd patterns
based on the most highly supported scenario in structure  = −  <dig> .0; see additional files  <dig> and 4), the means of ancestry proportions were declining from south to north, whereas the opposite was true for the standard deviations .
as expected, samples from the northern distribution had the lowest numbers of private alleles on either continent .
moreover, iceland had the highest average level in observed heterozygosities .
upon correcting for the presence of null alleles using the ina method  <cit>  in freena, similar results were obtained, albeit with different absolute levels.
again, two populations were clearly statistically supported based on the ∆k statistics for the structure output  = −  <dig> .0; see additional file 5), and the same geographic cline was apparent over the whole distribution range of the north atlantic eels .
the geographic groupings were statistically robust as revealed by pairwise non-parametric tests.
based on the inferred ancestry proportions in structure, significant differentiation was apparent among four out of the six comparisons among groups within continents.
in europe, differentiation was apparent among southern and northern european samples before and after boxcox transformation , and the same trend was found between the intermediate ranges and the southern locations .
the intermediate ranges were only marginally different from the northern locations .
among american eels, both southern and northern groups were statistically different from the intermediate group after boxcox transformation .
in line with the geographic admixture cline, we identified members of the southern-most groups of each continent as representing the purest populations: the american population from river st johns, florida, and the european population from river minho, portugal, respectively.
these samples were used as baseline populations for our f <dig> hybrid simulation approach.
importantly, the simulation of an ibd pattern in the european eel revealed increasing values of both ibd correlation coefficients and significance levels of the mantel tests  when increasing levels of gene flow were applied.
assuming that the real ibd pattern  <cit>  was solely explained by the f <dig> hybridization cline, an average f <dig> proportion of ca.
15% among populations with a maximum of 30% in icelandic eels can explain the ibd signal detected.
whilst a linear regression fit the data best for correlation coefficients r , exponential curve fitting performed slightly better for the slopes of the trend lines b .
it might be also worth mentioning that positive ibd correlations largely outnumbered flat or negative correlations  in the literature; a trend which might have some biological meaning.
however, these numbers exceed by far the literature values reported on clearly evidenced first generation hybrids both at continental ranges  <cit>  as well as in the vicinity of the sargasso sea  <cit> .
thus, our simulation results are not fully consistent with observation of hybrids in nature, and it seems that our assumptions of increasing clines of f <dig> hybrids alone represents a clear oversimplification.
hence, in a second, more realistic attempt, we performed a new set of simulations accounting for the observed diversity of admixture values derived from structure .
following this method we did not produce f <dig> genotypes with intermediate admixture values, but admixed genotypes satisfying admixture values of near to qexpected =  <dig>  .
as a result we were able to recapitulate the same observation and pattern as in analysis  <dig> .
finally, assuming an im model as implemented in ima <dig>  we were able to refute the possibility that incomplete lineage sorting and thus ancient polymorphism skewed our data.
we then directly estimated the reciprocal migration rates among the two atlantic eel species.
chain convergence was achieved in most cases and three independent runs per population pair gave very similar, and thus reproducible results .
moreover, posterior probability distributions of the population size and migration rate parameters showed one clear peak with fairly narrow ranges .
therefore, although estimated values must be considered with caution, we can reasonably draw some conclusion from these results.
importantly, the data confirm our already observed clinal admixture trend with high migration rates between northern populations and subsequent decreases toward southern pairs .
the results indicate a ~ <dig> to 10-fold difference in gene flow with a major contribution from american toward european eels, and this for all three continental population pairs .
it is worth mentioning that our nine markers have different discriminatory power in detecting ibd as depicted by the variation in locus-specific f
st
values .
however, this variation is unlikely to have interfered with our ability to detect hybridization as depicted by the clear results of our various simulation approaches.
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parameters are estimated from the location of the peaks of the estimated posterior probability densities.
population size and migration rate parameters are scaled by the mutation rate μ. aro, a. rostrata; aan, a. anguilla; ni, the effective size of population i; μ, the mutation rate; m, the migration rate per generation per copy.
discussion
the fascinating life history of atlantic eels that involves a rare catadromous spawning migration, their economic impact and the concomitant international trade has prompted numerous studies in the past 30 years.
therefore it is astonishing that, despite the large efforts made, so many questions still remain unanswered  <cit> .
our empirical and simulated data suggest that the paradigm of panmixia that was recently wavering due to major population genetics advances might still hold  <cit> .
here we propose a scenario consisting of two randomly mating populations with fluctuating and clinal introgression rates; this result alone will affect the large-scale management of both endangered species since first- and later generation hybrids apparently accumulate at northern habitats.
hybridization pattern among north-atlantic eels
in our attempt to detect hybrids relying on nine microsatellite markers, we came to two major conclusions.
first,  <dig> icelandic eels sharing an american mtdna are genetically intermediate  and significantly depart from pure european or american samples .
second, the admixture clines observed on both sides of the atlantic  are very unlikely to be obtained by chance alone.
combinational statistics tells us that obtaining a hierarchic rank of  <dig>   <dig>   <dig> from three samples  has a probability p = ; obtaining a rank of  <dig>   <dig>   <dig>   <dig> from  <dig> samples  has a probability p = .
therefore by combining these occurrences, we have only one chance in  <dig>  of obtaining the result shown in figure 4a.
though, one cannot fully exclude the presence of homoplasy, noise did not erase genetic information as previously assumed  <cit> .
here we argue that latitudinal clines of admixed individuals between european and american eels might be sufficient to explain ibd patterns observed in the european eel using neutral microsatellite markers  <cit> .
the latitudinal hybrid clines are most likely due to a very recent onset of gene flow after a secondary overlap and could reflect superior hybrid fitness in the northern parts of the atlantic.
the following line of argument supports this inference.
first, we confirm that icelandic samples display high admixture proportions when compared to the continental samples.
hybrid frequencies in iceland are high and the proportion of inferred american haplotypes is in good agreement with reported cyto-nuclear disequilibria  <cit> , which is a hallmark of hybrid zones  <cit> .
second, while mitochondrial lineages in eels remain 100% distinct on both sides of the atlantic, the hybridization signal expands further to continental stocks in the nuclear genes, with decreasing latitudinal allelic richness and admixture proportions.
thus, this diffusion most likely depicts recent on-going gene flow introducing new alleles into each continental nuclear gene pool by back-crossing in the absence of maternal lineage mixing.
third, when simulating declining proportions of both f <dig> hybrids or more realistic fractions of admixed individuals from north to south, a stepwise decrease of approximately 5% of foreign alleles per  <dig>  km of coastline would explain the ibd signal reported in wirth and bernatchez  <cit> , whereas absent or low gene flow will fail to do so.
fourth, an isolation-with-migration model fits the data better then the alternative hypothesis of clinal ancestral polymorphism due to incomplete lineage sorting.
the latter analysis also provides strong evidence for directional gene flow from american toward european eels, which is in good agreement with the absence of isolation-by-distance in the american eel  <cit> .
importantly, previous studies suggest that actual f <dig> and later generation hybrids are both rare on the continents  <cit>  as well as in the vicinity of the spawning grounds in the sargasso sea  <cit> .
thus, the portions of f <dig> hybrids needed to explain our finding are not fully consistent with observations in nature.
however, assuming a more realistic scenario, we could show that increasing latitudinal introgression clines impacted ibd in an identical way as using actual f <dig> hybrid clines.
this can be explained by the fact that the distribution of q-values of admixed individuals averages a value very close to the one apparent in f <dig> hybrids .
given semi-permeable barriers to gene flow  neutral markers that show intermediate linkage disequilibria with loci involved in reproductive isolation exhibit reduced gene flow compared to other neutral but unlinked markers.
such markers with reduced effective migration can display clining gene flow, as evidenced in tropical eel species which showed both admixture and introgression clines to produce an ibd pattern  <cit> .
while we did not explicitly distinguish between these two different clines in our simulations, we put forth strong evidence that gene flow among atlantic eel species influence measures of population structure such as ibd.
reunifying ibd and ibt signals
in light of our results, we are now in the position to consolidate some of the seemingly contradictory results of recent population genetics studies.
catchment areas for the earliest larval stages  are clearly overlapping among atlantic eels .
in contrast, the number of hybrids sampled in the sargasso sea was very low, and centred in the spawning areas of the european eel  <cit> .
thus, there are reasons to believe that actual spawning areas are partially “allopatric”, possibly due to strong oceanic fronts that separate spawning eels within distinct areas  <cit> .
interestingly, albert et al.
<cit>  showed that the proportion of hybrids reaching icelandic waters peaked in  <dig> and  <dig>  while it decreased from  <dig> to  <dig>  this trend could explain the strong patterns of isolation-by-distance in two independent studies that relied on samples from  <dig> to  <dig>  <cit> ; while later studies did not find support for population structure at all  <cit> .
the former could then be expected to lead to the clear pattern of isolation-by-time in genotype data that spans the temporal time-frame in question  <cit> .
thus, as a working hypothesis, sudden bursts of hybridization and hybrid arrivals could explain temporally unstable patterns of ibd, with a clear signal of ibt.
accordingly, the fluctuations in hybrid recruit proportions would reflect changes in the degree and timing of overlap of spawning grounds in an unstable oceanic environment  <cit> .
since only a very small percentage  of hatched eel larvae is expected to reach suitable habitats within two years of dispersal  <cit> , the lower the overlap of each species spawning grounds, the lower and less significant the correlation coefficients of the ibd signal on either continent would be, such as found in  <cit> , or  <cit> .
a recent modelling approach has highlighted that even minimal mixing among any existing sub-populations in the spawning grounds would entirely erase detectable signatures of genetic divergence in arriving eel recruits at the continents  <cit> .
thus, among the alternative explanations they offer, we assume that admixture and introgression clines can completely explain our findings.
evidence for cyto-nuclear incompatabilites and asymmetric gene flow
even though iceland is at the intersect of the north atlantic eel distributions, icelandic eels show a clear affinity towards the european eel gene pool: the suspect individuals carrying american haplotypes in iceland are hybrids, and no pure american migrants were recovered there.
this observation is in accordance with the results collected by albert et al.
<cit>  who could not identify pure american eels in iceland based on a much larger sample size and denser geographic coverage.
recently, frankowski et al.
<cit>  reported the regional sampling of eels with american mitochondria in europe.
however, these eels were sampled from both aquacultures and frequently stocked german rivers.
in the absence of nuclear genetic evidence, these eels were most likely introduced american eels, which escaped into natural habitats, reminiscent of the situation in japan, where imported foreign eels are frequently detected in rivers  <cit> .
if anything, we would assume that stocking activities would rather erase our fine signal of clinal gene flow.
while american and european eels are clearly separated according to mitochondrial data, interbreeding signatures gradually increased toward northern habitats in our data.
this cyto-nuclear disequilibrium had already been noticed earlier by avise et al.
<cit> .
importantly, using an rna-seq approach, gagnaire and colleagues  <cit>  delivered a very intriguing molecular mechanism that could explain this finding, and has implications for asymmetric inter-species hybridization.
two components of the atp synthase complex, one encoded in the mitochondrium , the other in the nucleus , show significant signs of diversifying selection between the two atlantic eel species possibly due to co-adaptation in at least one of the species.
thus, conflicts between certain cyto-nuclear combinations could render energy metabolism dysfunctional, diminishing the number of possible hybrid combinations as a result.
interestingly, the authors deliver strong evidence for asymmetric gene flow in several nuclear genes, most of which are directed into the european gene pool.
this observation is matching our data based on neutral microsatellite markers.
dispersal time and development
the most obvious difference between the two north atlantic eel species is their divergent larval dispersal strategy, coupled with differences in the onset of metamorphosis  <cit> .
data for the timing of metamorphosis and recruitment are based on otolith daily increments  <cit> .
however, these calculations provided contrasting results between research groups, and, even more worrying, “back-calculated” larval migration times did not match the field observations  <cit> .
these inconsistencies suggest that daily increments are not suitable for calculating the timing of dispersal, but rather that they provide a descent proxy for the timing of metamorphosis of leptocephali into glass eels, which takes place on the verge of open ocean and continental shelves  <cit> .
kettle and haines  <cit>  predicted a minimum of two years for larvae to successfully cross the atlantic solely by passive drift, which was concordant with the early estimates based on larval growth rates by schmidt  <cit> .
this would suggest a three- to six-fold longer migration time for the european eel compared to its american congener , which arrives within some 7–12 months .
that fact might explain why hybrid eels, are also most abundant at intermediate ranges .
hybrid fitness and ecological peculiarities in iceland
in light of the frequent detection of hybrids in iceland, we speculate on the possibility that nordic habitats, especially iceland and greenland  <cit>  might depict ecotone habitats compared to the much warmer continents.
freshwater habitats in these areas were definitely uninhabitable during extensive glaciation events in the pleistocene and must have been colonized afterward .
it is suggested that environmental peculiarities characterize eel habitats in iceland, as freshwater temperatures are typically much lower there as compared to most potential continental habitats  <cit> .
this anomaly is directly reflected in icelandic glass eel’s otolith microstructure and microchemistry, which lack both the usual sharp decrease in sr:ca ratios and elevated increment accumulations  <cit> .
in addition, it is worth noting that the diffuse otolith increment zone after metamorphosis has never been observed in any other eel species outside iceland.
thus, environmental opportunity might favour f <dig> hybrids in iceland and other nordic habitats.
in support of this argument, albert et al.
<cit>  quantified the hybrid proportions in both recruiting and resident eel stages over several years.
a total of 70% of putative hybrids fell into the first generation category, whereas 30% belonged to later generation hybrids.
they observed an approximately two-fold increase in hybrid proportions from the recruiting glass eel to the resident yellow eel stages.
these results suggest a higher hybrid survival upon residency.
the presence of second and later generation hybrids indeed demonstrates that hybrids transmitted their genes to the next generations and this would explain why the admixture extends further south on both continents.
final remarks
the calculations presented here are based on the strongest ibd detected and therefore represent the highest gene flow estimates that could be extrapolated from the genetic data.
the age-dependent fitness differences  <cit>  were not taken into account here.
therefore, the hybridization rate is also somewhat biased, given that the northern a. anguilla samples in the analysed data set are collected from yellow eels fin clips and not from glass eels.
however, as has been mentioned elsewhere  <cit> , temporal inter-cohort variation is not expected to lead to a clinally structured partition of genetic variation, but rather to unordered differentiation patterns.
the rather weak traces of admixture detected by albert et al.
<cit>  in the continental samples only moderately fit with our results.
however, it is worth noting that the few american eels with a later generation hybrid signature belonged to the most northern samples  and as such follow the cline presented here.
moreover, als et al.
<cit>  only detected one hybrid leptocephalus out of  <dig> eel larvae in the sargasso sea.
this very low hybrid prevalence is strikingly different from what is observed at later developmental stages in iceland  <cit> .
therefore, selection gradients and landscape genetics must be the main evolutionary forces shaping the latitudinal cline.
leaving aside the statistical issues , the nature of the genetic markers , their relative merits and the quantitative aspects, we now have a couple of clear scenarios and hypotheses to test.
the fast developing next-generation sequencing field has already provided us with primer molecular data, that open up the opportunity to enlighten the ominous marine part of the eel’s life cycle .
our understanding of the evolutionary dynamics of eel stocks and the selective factors that shape introgressive hybridization in north-atlantic eels is thus currently going through a quantum leap.
especially if the northern distribution range can be more systematically and extensively sampled from now on, we will be able to appreciate the actual rate of hybridization among the two atlantic eel species and follow its fluctuation in space and time.
this would also have important practical implications in the monitoring of conservation strategies that have been enacted in regulation  no 1100/ <dig> by the european union  <cit>  in response to the dramatic decline in eel recruitment in the past decades  <cit> .
as a prerequisite for appropriate actions, the fact that admixed individuals and hybrids preferentially tend to accumulate in northern habitats must be accounted for.
for example, bursts of increased inter-species hybridization in certain years might lead to increased numbers of northward-bound recruits, and reduced catchment in more southern ranges.
CONCLUSIONS
here, we explicitly focused on the influence of hybridization on genetic differentiation signatures in north atlantic eels.
we relied on the available nuclear microsatellite genotypes and mitochondrial sequence data of  <dig>  samples from both continental american and european eels as well as from intermediate iceland.
when simulating continuously increasing proportions of f <dig> hybrid individuals from the southern to the northern-most locations in europe, highly significant isolation-by-distance patterns arose, that are reminiscent of previously published data  <cit> .
therefore, introgressive hybridization alone is sufficient to explain the correlation of geographic and genetic distances reported for the european freshwater eel.
moreover, contrasting signals among nuclear and mitochondrial lineages suggest a recent onset of gene flow, most likely after glacial retreat following the last ice age .
importantly, our findings are in agreement with previous results on genetic isolation patterns in european eels, either based on geography , or inter-annual genetic composition .
if we can assume that the known overlap of the two species’ spawning grounds  <cit>  is annually changing, the ibd signal should decline in some years , and increase in others.
this will then automatically lead to an even higher ibt signal among annual recruitment waves within species.
the clear separation of mitochondrial lineages might be explained by cyto-nuclear disequilibria recently detected  <cit> .
thus, the lack of pure american expatriates in iceland , could be due to the american eel’s much faster ontogenetic development and metamorphosis which might prevent its settlement in this northern region .
all in all, evidence for hybrid survival in iceland  <cit>  favours the introgression hypothesis followed by subsequent backcrossing.
this might not only hold for iceland but for other nordic regions as well, and might generate a north-to-south-hybrid gradient in both atlantic eel species.

 hybridization, the interbreeding of diagnosably divergent species, is a major focus in evolutionary studies as it is a key concept for understanding the demographic and evolutionary cohesiveness of natural populations. hybridization has great potential to rapidly introduce variability into a recipient population, if barriers to recombination can be overcome. cooper put forth strong experimental evidence that sexual recombination speeds up the origin and spread of adaptations in an artificial environment compared to purely clonal strains of escherichia coli, thus supporting the theoretical predictions from the fisher-muller model. based on the evidence gathered from empirical data, including invasive species and signatures of massive horizontal gene transfer in a paradigmatic long-term asexual species, it might be generalized that in order to quickly adapt, any mechanism of lateral gene transfer or recombination is highly favored in unstable or novel environments. importantly, the same might apply for populations overlapping after a phase of allopatry, as is assumed to be the case for north atlantic eels. the two north atlantic eel species, anguilla anguilla and a. rostrata both have a remarkable catadromous life-cycle that comprises two long-range migrations through the open ocean, a continental growing phase, and a spawning stage in the sargasso sea. despite the key importance of the marine phase, most of the scientific investigations focused on the continental phase, and only few data exist on the oceanic parts of the eel’s life-history. to this day, migration routes and exact spawning sites of eels remain largely hypothetical. fifty years ago, to the great surprise of the scientific community, tucker suggested that the european eel should be considered an evolutionary dead-end of non-spawning individuals. however, with the advent of new molecular markers the genealogical distinctness of the two species was largely clarified. deligny and pantelouris and avise et al. argued for the continuation of the two-species-status, originally proposed by schmidt. this scenario was reinforced when mitochondrial genome sequences became available. recently, using miniaturized pop-up satellite archival transmitters, a small number of eels could be followed during their spawning migration off the european continental shelf towards the canary and azores current system. the authors confirmed the eels’ daily vertical migrations between depths of 200–1000 m at a speed of five to 25 km per day. *reconsidered by koehn: listed markers agree with mendelian inheritance and have objectively interpretable banding patterns. **statistically re-evaluated by applying χ2-statistics with correct degrees of freedom to test for hardy-weinberg-equilibrium. atlantic eel stocks have rapidly declined by over 95% compared to the levels prior to. as a consequence the european eel has been listed as critically endangered on the iucn red list. this dramatic decrease is attributable to a combination of factors, including habitat destruction, pollution, and over-fishing, climate induced changes in the gulf stream circulation, as well as the recent introduction and spread of anguillicola crassus, an exotic swimbladder nematode that was initially introduced along with infected japanese eels. disentangling the population structure of both north atlantic eel species thus has implications on international eel stock management both from conservation and fisheries perspectives. advanced molecular tools permitted the collection of larger and more sensitive data sets at the population level. however, despite huge sampling efforts these studies have come to different and even contradictory conclusions. three independent studies reported isolation-by-distance in a. anguilla which provided evidence against the long-held paradigm of panmixia for this species based on the measurement of very low, but nevertheless highly significant genetic structure within european eels. wirth and bernatchez suggested that ibd patterns are due to a stable temporal delay of spawning migration in eels from northern habitats, as distances are markedly extended compared to western and southern european populations. alternatively, more than one spawning area might be used by different populations and, also, different currents might carry the leptocephali larvae back from the sargasso sea to their parent’s original freshwater habitats in europe. thirdly, albeit seemingly less likely, assortative mating among regional groups might be responsible for the observed ibd patterns. finally, maes et al. suggested that, given the random factors affecting spawning success in the open ocean, a sweepstake strategy might explain genetically patchy recruitment in sampling locations across europe, leading to a weak but significant isolation-by-time signal. however, most recent studies did not find statistical support for population structure and rejected both ibd and ibt altogether, thus favoring panmixia in both atlantic eel species. in addition, temporal variation among recruits between different arrival waves within the same years significantly exceeded both the geographic and inter-annual genetic differentiation reported above. several points have been raised to explain the discrepancies between early evidence against panmixia and later studies. palm et al. argued that genetic differentiation in the european eel might be largely explained by uncontrolled temporal variation between juvenile glass eel samples. dannewitz et al. speculated that the use of different cohorts or life stages in the initial studies with strongest support against panmixia could have lead to artifactual isolation-by-distance patterns. lastly, after evaluating the weak genetic differentiation among north atlantic eels using basic summary statistics, mank and avise concluded that the large overlap in their allelic frequencies is generated by extensive homoplasy associated with a mutation-driven saturation effect. this argument casted doubts on the usefulness of rapidly evolving microsatellite loci for short term evolutionary and hybridization studies in eels. while there exists strong empirical evidence for near panmixia in both species of north atlantic eels, not all questions could be successfully addressed. first, it is difficult to see how saturated markers and patchy cohorts within sampling locations could generate statistically significant patterns of ibd over the whole distributional range. second, the same markers detected the highest signatures of admixture in icelandic samples are consistent with meristic and genetic data that suggested the existence of hybrids in northern eel habitats in general. this pattern suggests the existence of a hybrid zone in the atlantic that could lead to clines of admixture in both species of eel. in fact, this hypothesis has recently been invigorated by a study that relied on an integrated demographic-genetic model and explicitly accounted for the different levels of larval and adult mixing during oceanic migrations. this model showed that even minimal levels of mixing among initially separate sub-populations during both larval dispersal or adult migration are sufficient to entirely erase any genetic differences among them. building on this finding, the authors offer two explanations for why the geographic differentiation component might have been overestimated in the past: a limited number of temporal recruits analysed, introgression through inter-species hybridization and non-random dispersal. there is a growing number of studies that show hybridization with molecular data, but no study to date has systematically tested this in the north atlantic eels across their entire range of distribution. traditionally, many zoologists are reluctant to consider hybridization as an important evolutionary process that generates new species, since the pre- and post-mating barriers to establishing f hybrids are often considerable. however, there is a growing number of examples of reticulate evolution in nature, particularly from plants and fungi. a very well known feature of hybrid zones are coincidental changes at several independent characters, resulting in parallel frequency gradients, and such a linkage is measurably exemplified in north atlantic eel species, a. anguilla and a. rostrata. here, cyto-nuclear disequilibrium has been identified in the narrow zone of species overlap in iceland, which scales well with a transition zone based on vertebral counts, a nearly diagnostic trait between the two species of eels. more specifically, avise et al. estimated the hybrid fraction in iceland to be to 4%. later, albert et al. evaluated the extent of hybridization and tested for the occurrence of hybrids beyond the first generations, using aflp markers. a total hybrid fraction of % was identified, of which 30% were considered to be later generation hybrids. h n h, mitochondrial frequency of a. rostrata haplotypes in iceland; n, sampling size. here, we investigate the extent of population genetic differentiation, and by performing simulations, ask whether ibd patterns might be produced by increasing inter-species hybridization and admixture clines. such a scenario would also be able to explain the emergence of an ibt signal and fluctuating genetic structure signatures over time. by combining and extending two available data sets genotyped at nine microsatellite markers, we investigated eels sampled from the whole north atlantic distribution area for signals of inter-species hybridization. in a first step, we inferred the admixture proportions for each individual and tested if the most extreme ibd signal detected in european eels can be explained by latitudinal admixture and introgression clines using a simulation approach. second, we quantified the amount of gene flow that would be necessary to generate the observed correlations, and, finally, we tested if incomplete lineage sorting or recent introgression are most likely to explain the observed lack of monophyly in most nuclear markers observed so far.  summary statistics nine microsatellite loci were used to infer intra-specific relationships among north-atlantic eels. inter-specific comparison of genetic differentiation for both fst and rst estimates were highly congruent with one another at and, respectively. thus, microsatellite markers used appear to essentially evolve by addition or removal of simple repeats in a step-wise manner. overall, levels of observed polymorphism were high in north atlantic eels, ranging from ho =  at locus aro to ho =  at locus ang with a mean of ho = . the same was true for the average numbers of alleles when correcting for sample sizes, ranging from ar =  in aro to ar =  at locus ang none of the pairwise tests for linkage disequilibrium among loci was significant after bonferroni correction. however, micro-checker revealed the presence of null alleles for the two newly added loci ang and aro this observation might explain the moderately high fis values for most sampling localities and a systematic deviation from hwe for all populations. however, after correction for null alleles using the algorithm freena, all but three out of sampling locations were compatible with hwe after bonferroni correction and in each case where a significant deviation from hwe was detected, only a single locus contributed to the effect. detection of hybrid eels in iceland based on diagnostic restriction digests of cytb fragments, out of icelandic eels carried american haplotypes, whereas none of the continental north atlantic eels showed restriction patterns corresponding to the other species. thus, as already described, there appears to be a rather clear-cut sorting of mitochondrial lineages with the species boundaries. the divergence was less pronounced based on ancestry proportions at nuclear markers, which consistently separated american and european gene pools for the most likely number of populations, k =  importantly, the average ancestry proportion of the “suspects” in iceland based on nine microsatellite markers was intermediate compared to those generated from blind draws of random individuals from either species, and thus these suspected mtdna hybrid individuals most likely represent true f hybrids. in addition, the mean ancestry proportion of non-suspect icelandic eels was not significantly different from the european mean, albeit slightly shifted toward american eels. when adding prior geographic information on continental stocks, no american expatriate was detected in iceland, but two eels carrying a european mtdna haplotype were assigned as pure american given their nuclear data. geographic admixture and introgression clines and their impact on ibd patterns based on the most highly supported scenario in structure  = −.0; see additional files and 4), the means of ancestry proportions were declining from south to north, whereas the opposite was true for the standard deviations. as expected, samples from the northern distribution had the lowest numbers of private alleles on either continent. moreover, iceland had the highest average level in observed heterozygosities. upon correcting for the presence of null alleles using the ina method in freena, similar results were obtained, albeit with different absolute levels. again, two populations were clearly statistically supported based on the ∆k statistics for the structure output  = −.0; see additional file 5), and the same geographic cline was apparent over the whole distribution range of the north atlantic eels. the geographic groupings were statistically robust as revealed by pairwise non-parametric tests. based on the inferred ancestry proportions in structure, significant differentiation was apparent among four out of the six comparisons among groups within continents. in europe, differentiation was apparent among southern and northern european samples before and after boxcox transformation, and the same trend was found between the intermediate ranges and the southern locations. the intermediate ranges were only marginally different from the northern locations. among american eels, both southern and northern groups were statistically different from the intermediate group after boxcox transformation. in line with the geographic admixture cline, we identified members of the southern-most groups of each continent as representing the purest populations: the american population from river st johns, florida, and the european population from river minho, portugal, respectively. these samples were used as baseline populations for our f hybrid simulation approach. importantly, the simulation of an ibd pattern in the european eel revealed increasing values of both ibd correlation coefficients and significance levels of the mantel tests when increasing levels of gene flow were applied. assuming that the real ibd pattern was solely explained by the f hybridization cline, an average f proportion of ca. 15% among populations with a maximum of 30% in icelandic eels can explain the ibd signal detected. whilst a linear regression fit the data best for correlation coefficients r, exponential curve fitting performed slightly better for the slopes of the trend lines b. it might be also worth mentioning that positive ibd correlations largely outnumbered flat or negative correlations in the literature; a trend which might have some biological meaning. however, these numbers exceed by far the literature values reported on clearly evidenced first generation hybrids both at continental ranges as well as in the vicinity of the sargasso sea. thus, our simulation results are not fully consistent with observation of hybrids in nature, and it seems that our assumptions of increasing clines of f hybrids alone represents a clear oversimplification. hence, in a second, more realistic attempt, we performed a new set of simulations accounting for the observed diversity of admixture values derived from structure. following this method we did not produce f genotypes with intermediate admixture values, but admixed genotypes satisfying admixture values of near to qexpected = . as a result we were able to recapitulate the same observation and pattern as in analysis. finally, assuming an im model as implemented in ima we were able to refute the possibility that incomplete lineage sorting and thus ancient polymorphism skewed our data. we then directly estimated the reciprocal migration rates among the two atlantic eel species. chain convergence was achieved in most cases and three independent runs per population pair gave very similar, and thus reproducible results. moreover, posterior probability distributions of the population size and migration rate parameters showed one clear peak with fairly narrow ranges. therefore, although estimated values must be considered with caution, we can reasonably draw some conclusion from these results. importantly, the data confirm our already observed clinal admixture trend with high migration rates between northern populations and subsequent decreases toward southern pairs. the results indicate a ~ to 10-fold difference in gene flow with a major contribution from american toward european eels, and this for all three continental population pairs. it is worth mentioning that our nine markers have different discriminatory power in detecting ibd as depicted by the variation in locus-specific f st values. however, this variation is unlikely to have interfered with our ability to detect hybridization as depicted by the clear results of our various simulation approaches. aro aan aro to aan aan to aro aro to aan aan to aro parameters are estimated from the location of the peaks of the estimated posterior probability densities. population size and migration rate parameters are scaled by the mutation rate μ. aro, a. rostrata; aan, a. anguilla; ni, the effective size of population i; μ, the mutation rate; m, the migration rate per generation per copy. discussion the fascinating life history of atlantic eels that involves a rare catadromous spawning migration, their economic impact and the concomitant international trade has prompted numerous studies in the past 30 years. therefore it is astonishing that, despite the large efforts made, so many questions still remain unanswered. our empirical and simulated data suggest that the paradigm of panmixia that was recently wavering due to major population genetics advances might still hold. here we propose a scenario consisting of two randomly mating populations with fluctuating and clinal introgression rates; this result alone will affect the large-scale management of both endangered species since first- and later generation hybrids apparently accumulate at northern habitats. hybridization pattern among north-atlantic eels in our attempt to detect hybrids relying on nine microsatellite markers, we came to two major conclusions. first, icelandic eels sharing an american mtdna are genetically intermediate and significantly depart from pure european or american samples. second, the admixture clines observed on both sides of the atlantic are very unlikely to be obtained by chance alone. combinational statistics tells us that obtaining a hierarchic rank of from three samples has a probability p =; obtaining a rank of from samples has a probability p =. therefore by combining these occurrences, we have only one chance in of obtaining the result shown in figure 4a. though, one cannot fully exclude the presence of homoplasy, noise did not erase genetic information as previously assumed. here we argue that latitudinal clines of admixed individuals between european and american eels might be sufficient to explain ibd patterns observed in the european eel using neutral microsatellite markers. the latitudinal hybrid clines are most likely due to a very recent onset of gene flow after a secondary overlap and could reflect superior hybrid fitness in the northern parts of the atlantic. the following line of argument supports this inference. first, we confirm that icelandic samples display high admixture proportions when compared to the continental samples. hybrid frequencies in iceland are high and the proportion of inferred american haplotypes is in good agreement with reported cyto-nuclear disequilibria, which is a hallmark of hybrid zones. second, while mitochondrial lineages in eels remain 100% distinct on both sides of the atlantic, the hybridization signal expands further to continental stocks in the nuclear genes, with decreasing latitudinal allelic richness and admixture proportions. thus, this diffusion most likely depicts recent on-going gene flow introducing new alleles into each continental nuclear gene pool by back-crossing in the absence of maternal lineage mixing. third, when simulating declining proportions of both f hybrids or more realistic fractions of admixed individuals from north to south, a stepwise decrease of approximately 5% of foreign alleles per  km of coastline would explain the ibd signal reported in wirth and bernatchez, whereas absent or low gene flow will fail to do so. fourth, an isolation-with-migration model fits the data better then the alternative hypothesis of clinal ancestral polymorphism due to incomplete lineage sorting. the latter analysis also provides strong evidence for directional gene flow from american toward european eels, which is in good agreement with the absence of isolation-by-distance in the american eel. importantly, previous studies suggest that actual f and later generation hybrids are both rare on the continents as well as in the vicinity of the spawning grounds in the sargasso sea. thus, the portions of f hybrids needed to explain our finding are not fully consistent with observations in nature. however, assuming a more realistic scenario, we could show that increasing latitudinal introgression clines impacted ibd in an identical way as using actual f hybrid clines. this can be explained by the fact that the distribution of q-values of admixed individuals averages a value very close to the one apparent in f hybrids. given semi-permeable barriers to gene flow neutral markers that show intermediate linkage disequilibria with loci involved in reproductive isolation exhibit reduced gene flow compared to other neutral but unlinked markers. such markers with reduced effective migration can display clining gene flow, as evidenced in tropical eel species which showed both admixture and introgression clines to produce an ibd pattern. while we did not explicitly distinguish between these two different clines in our simulations, we put forth strong evidence that gene flow among atlantic eel species influence measures of population structure such as ibd. reunifying ibd and ibt signals in light of our results, we are now in the position to consolidate some of the seemingly contradictory results of recent population genetics studies. catchment areas for the earliest larval stages are clearly overlapping among atlantic eels. in contrast, the number of hybrids sampled in the sargasso sea was very low, and centred in the spawning areas of the european eel. thus, there are reasons to believe that actual spawning areas are partially “allopatric”, possibly due to strong oceanic fronts that separate spawning eels within distinct areas. interestingly, albert et al. showed that the proportion of hybrids reaching icelandic waters peaked in and while it decreased from to this trend could explain the strong patterns of isolation-by-distance in two independent studies that relied on samples from to; while later studies did not find support for population structure at all. the former could then be expected to lead to the clear pattern of isolation-by-time in genotype data that spans the temporal time-frame in question. thus, as a working hypothesis, sudden bursts of hybridization and hybrid arrivals could explain temporally unstable patterns of ibd, with a clear signal of ibt. accordingly, the fluctuations in hybrid recruit proportions would reflect changes in the degree and timing of overlap of spawning grounds in an unstable oceanic environment. since only a very small percentage of hatched eel larvae is expected to reach suitable habitats within two years of dispersal, the lower the overlap of each species spawning grounds, the lower and less significant the correlation coefficients of the ibd signal on either continent would be, such as found in, or. a recent modelling approach has highlighted that even minimal mixing among any existing sub-populations in the spawning grounds would entirely erase detectable signatures of genetic divergence in arriving eel recruits at the continents. thus, among the alternative explanations they offer, we assume that admixture and introgression clines can completely explain our findings. evidence for cyto-nuclear incompatabilites and asymmetric gene flow even though iceland is at the intersect of the north atlantic eel distributions, icelandic eels show a clear affinity towards the european eel gene pool: the suspect individuals carrying american haplotypes in iceland are hybrids, and no pure american migrants were recovered there. this observation is in accordance with the results collected by albert et al. who could not identify pure american eels in iceland based on a much larger sample size and denser geographic coverage. recently, frankowski et al. reported the regional sampling of eels with american mitochondria in europe. however, these eels were sampled from both aquacultures and frequently stocked german rivers. in the absence of nuclear genetic evidence, these eels were most likely introduced american eels, which escaped into natural habitats, reminiscent of the situation in japan, where imported foreign eels are frequently detected in rivers. if anything, we would assume that stocking activities would rather erase our fine signal of clinal gene flow. while american and european eels are clearly separated according to mitochondrial data, interbreeding signatures gradually increased toward northern habitats in our data. this cyto-nuclear disequilibrium had already been noticed earlier by avise et al.. importantly, using an rna-seq approach, gagnaire and colleagues delivered a very intriguing molecular mechanism that could explain this finding, and has implications for asymmetric inter-species hybridization. two components of the atp synthase complex, one encoded in the mitochondrium, the other in the nucleus, show significant signs of diversifying selection between the two atlantic eel species possibly due to co-adaptation in at least one of the species. thus, conflicts between certain cyto-nuclear combinations could render energy metabolism dysfunctional, diminishing the number of possible hybrid combinations as a result. interestingly, the authors deliver strong evidence for asymmetric gene flow in several nuclear genes, most of which are directed into the european gene pool. this observation is matching our data based on neutral microsatellite markers. dispersal time and development the most obvious difference between the two north atlantic eel species is their divergent larval dispersal strategy, coupled with differences in the onset of metamorphosis. data for the timing of metamorphosis and recruitment are based on otolith daily increments. however, these calculations provided contrasting results between research groups, and, even more worrying, “back-calculated” larval migration times did not match the field observations. these inconsistencies suggest that daily increments are not suitable for calculating the timing of dispersal, but rather that they provide a descent proxy for the timing of metamorphosis of leptocephali into glass eels, which takes place on the verge of open ocean and continental shelves. kettle and haines predicted a minimum of two years for larvae to successfully cross the atlantic solely by passive drift, which was concordant with the early estimates based on larval growth rates by schmidt. this would suggest a three- to six-fold longer migration time for the european eel compared to its american congener, which arrives within some 7–12 months. that fact might explain why hybrid eels, are also most abundant at intermediate ranges. hybrid fitness and ecological peculiarities in iceland in light of the frequent detection of hybrids in iceland, we speculate on the possibility that nordic habitats, especially iceland and greenland might depict ecotone habitats compared to the much warmer continents. freshwater habitats in these areas were definitely uninhabitable during extensive glaciation events in the pleistocene and must have been colonized afterward. it is suggested that environmental peculiarities characterize eel habitats in iceland, as freshwater temperatures are typically much lower there as compared to most potential continental habitats. this anomaly is directly reflected in icelandic glass eel’s otolith microstructure and microchemistry, which lack both the usual sharp decrease in sr:ca ratios and elevated increment accumulations. in addition, it is worth noting that the diffuse otolith increment zone after metamorphosis has never been observed in any other eel species outside iceland. thus, environmental opportunity might favour f hybrids in iceland and other nordic habitats. in support of this argument, albert et al. quantified the hybrid proportions in both recruiting and resident eel stages over several years. a total of 70% of putative hybrids fell into the first generation category, whereas 30% belonged to later generation hybrids. they observed an approximately two-fold increase in hybrid proportions from the recruiting glass eel to the resident yellow eel stages. these results suggest a higher hybrid survival upon residency. the presence of second and later generation hybrids indeed demonstrates that hybrids transmitted their genes to the next generations and this would explain why the admixture extends further south on both continents. final remarks the calculations presented here are based on the strongest ibd detected and therefore represent the highest gene flow estimates that could be extrapolated from the genetic data. the age-dependent fitness differences were not taken into account here. therefore, the hybridization rate is also somewhat biased, given that the northern a. anguilla samples in the analysed data set are collected from yellow eels fin clips and not from glass eels. however, as has been mentioned elsewhere, temporal inter-cohort variation is not expected to lead to a clinally structured partition of genetic variation, but rather to unordered differentiation patterns. the rather weak traces of admixture detected by albert et al. in the continental samples only moderately fit with our results. however, it is worth noting that the few american eels with a later generation hybrid signature belonged to the most northern samples and as such follow the cline presented here. moreover, als et al. only detected one hybrid leptocephalus out of eel larvae in the sargasso sea. this very low hybrid prevalence is strikingly different from what is observed at later developmental stages in iceland. therefore, selection gradients and landscape genetics must be the main evolutionary forces shaping the latitudinal cline. leaving aside the statistical issues, the nature of the genetic markers, their relative merits and the quantitative aspects, we now have a couple of clear scenarios and hypotheses to test. the fast developing next-generation sequencing field has already provided us with primer molecular data, that open up the opportunity to enlighten the ominous marine part of the eel’s life cycle. our understanding of the evolutionary dynamics of eel stocks and the selective factors that shape introgressive hybridization in north-atlantic eels is thus currently going through a quantum leap. especially if the northern distribution range can be more systematically and extensively sampled from now on, we will be able to appreciate the actual rate of hybridization among the two atlantic eel species and follow its fluctuation in space and time. this would also have important practical implications in the monitoring of conservation strategies that have been enacted in regulation no 1100/ by the european union in response to the dramatic decline in eel recruitment in the past decades. as a prerequisite for appropriate actions, the fact that admixed individuals and hybrids preferentially tend to accumulate in northern habitats must be accounted for. for example, bursts of increased inter-species hybridization in certain years might lead to increased numbers of northward-bound recruits, and reduced catchment in more southern ranges.  here, we explicitly focused on the influence of hybridization on genetic differentiation signatures in north atlantic eels. we relied on the available nuclear microsatellite genotypes and mitochondrial sequence data of samples from both continental american and european eels as well as from intermediate iceland. when simulating continuously increasing proportions of f hybrid individuals from the southern to the northern-most locations in europe, highly significant isolation-by-distance patterns arose, that are reminiscent of previously published data. therefore, introgressive hybridization alone is sufficient to explain the correlation of geographic and genetic distances reported for the european freshwater eel. moreover, contrasting signals among nuclear and mitochondrial lineages suggest a recent onset of gene flow, most likely after glacial retreat following the last ice age. importantly, our findings are in agreement with previous results on genetic isolation patterns in european eels, either based on geography, or inter-annual genetic composition. if we can assume that the known overlap of the two species’ spawning grounds is annually changing, the ibd signal should decline in some years, and increase in others. this will then automatically lead to an even higher ibt signal among annual recruitment waves within species. the clear separation of mitochondrial lineages might be explained by cyto-nuclear disequilibria recently detected. thus, the lack of pure american expatriates in iceland, could be due to the american eel’s much faster ontogenetic development and metamorphosis which might prevent its settlement in this northern region. all in all, evidence for hybrid survival in iceland favours the introgression hypothesis followed by subsequent backcrossing. this might not only hold for iceland but for other nordic regions as well, and might generate a north-to-south-hybrid gradient in both atlantic eel species. 
fluctuating degrees of introgressive hybridization between atlantic eel species are sufficient to explain temporally varying correlations of geographic and genetic distances reported for populations of the european eel. finally, fitting an isolation-with-migration model to our data supports the hypothesis of recent asymmetric introgression and refutes the alternative hypothesis of ancient polymorphism. hybridization, the interbreeding of diagnosably divergent species, is a major focus in evolutionary studies. by simulating increasing proportions of both f hybrids and admixed individuals from the southern to the northern-most locations, we were able to generate highly significant isolation-by-distance patterns in both cases, reminiscent of previously published data for the european eel. the detection of inter-species hybrids in iceland suggests on-going gene flow, but few studies to date have addressed the influence of introgression on genetic differentiation in north atlantic eels. eels, both from north america and europe migrate through the atlantic to mate in a vast, overlapping area in the sargasso sea. here, we show that while mitochondrial lineages remain completely distinct on both sides of the atlantic, limited hybridization is detectable with nuclear dna markers.
 hybridization, the interbreeding of diagnosably divergent species, is a major focus in evolutionary studies. eels, both from north america and europe migrate through the atlantic to mate in a vast, overlapping area in the sargasso sea. due to the lack of direct observation, it is unknown how these species remain reproductively isolated. the detection of inter-species hybrids in iceland suggests on-going gene flow, but few studies to date have addressed the influence of introgression on genetic differentiation in north atlantic eels.  here, we show that while mitochondrial lineages remain completely distinct on both sides of the atlantic, limited hybridization is detectable with nuclear dna markers. the nuclear hybridization signal peaks in the northern areas and decreases towards the southern range limits on both continents according to bayesian assignment analyses. by simulating increasing proportions of both f hybrids and admixed individuals from the southern to the northern-most locations, we were able to generate highly significant isolation-by-distance patterns in both cases, reminiscent of previously published data for the european eel. finally, fitting an isolation-with-migration model to our data supports the hypothesis of recent asymmetric introgression and refutes the alternative hypothesis of ancient polymorphism.  fluctuating degrees of introgressive hybridization between atlantic eel species are sufficient to explain temporally varying correlations of geographic and genetic distances reported for populations of the european eel. anguilla anguillaanguilla rostratagene flowisolation-by-distancesimulationmigration barriers 