BACKGROUND
many animals show complex life cycles organized into morphologically distinct phases separated by abrupt metamorphic transitions , as opposed to single static or continuously changing phases  <cit> .
complex life cycles are ubiquitous in nature and have evolved many times independently .
life stages are believed to represent alternative adaptations for optimal food and niche exploitation as well as conflicting tasks .
since darwin, evolutionary biologists have been interested in understanding the genetic associations between life cycle stages and to what extent discrete phases are free to evolve independently from one another.
metamorphosis marks drastic morphological, physiological, behavioural and ecological changes in the life cycles of animals  <cit> .
given the dramatic changes associated with metamorphosis, selection could differ before and after metamorphosis, and opposing selection might be more common than complimentary selection  <cit> .
in regard to the benefits of metamorphosis, the adaptive decoupling hypothesis  <cit>  predicts that traits separated by metamorphosis should be genetically uncorrelated, allowing distinct phases to respond independently to different selective forces, without correlated negative effects on traits of alternative phases.
studies testing the adaptive decoupling hypothesis have found contradictory results and the genetic associations between life cycle stages separated by metamorphosis remain poorly understood  <cit> .
our species of interest is the european eel , a facultative catadromous fish with a particularly complex life cycle that includes two metamorphoses.
after spawning in the remote sargasso sea, larvae of european eel are transported to the coasts of europe and north africa following the gulf stream and north atlantic current.
on reaching the continental shelf, eels undergo a first metamorphosis from larvae into glass eels , which complete their migration into continental feeding habitats as yellow eels.
after an extensive feeding/growing period, eels undergo a second metamorphosis into silver eels .
the so-called “silvering metamorphosis” encompasses morphological  as well as physiological modifications , together with the development of gonads.
these changes are beforehand preparation for the future spawning migration back to the sargasso sea, where eels reproduce and die  <cit> .
the european eel is an ideal species in which to study local selection.
first, it presents a large effective population size  that renders natural selection the major force determining genetic differences; hence the role of random genetic drift is expected to be negligible.
second, it is present across extremely heterogenous environments in terms of temperature , salinity , substrate, depth and productivity along its geographic distribution  <cit> .
despite such a wide distribution range, there is recent conclusive evidence for panmixia in european eel, i.e.
the existence of a single randomly mating population.
in the most comprehensive study to date genotyping over  <dig> individuals obtained throughout all the distribution range in europe at  <dig> microsatellite loci, als et al.
<cit>  showed a very low and nonsignificant genetic diffferentiation across europe  and a lack of substructuring among larvae collected in the sargasso sea .
moreover, no significant genetic differentiation was observed when comparing the samples obtained in europe vs. the larvae obtained from the spawning area .
panmixia was also confirmed at the genomic level in a study using a large dataset of >  <dig>  snps from  <dig> rad-sequenced european eels  <cit> , which showed low levels of genetic differentiation .
previous studies based on cohort analysis showed an unpatterned genetic heterogeneity  as samples did not group together according to sampling location or cohort, and consequently no pattern on isolation-by-distance or isolation-by-time was detected  <cit> .
if european eel larvae showed phylopatry to the parental original freshwater habitats, genetic differences would be expected across europe; hence, the lack of genetic structuring found suggest no larval homing and random larval migratory routes  <cit> .
one consequence of panmixia and random dispersal of larvae across habitats is that long-term local adaptation is not possible in eels, despite the high potential for selective responses due to high mortalities in both early and late life stages  <cit> .
any signature of spatially varying selection in a given generation is expected to be lost in the subsequent generation, preventing heritable trans-generational local adaptation  <cit> .
however, single-generation signatures of local selection are still detectable  <cit> .
studies of adaptive evolution in european eel have focused on the detection of signatures of local selection in glass eels.
using a panel of  <dig> coding-gene single nucleotide polymorphisms , ulrik et al.
<cit>  found signatures of selection at  <dig> loci in european eel, which constituted genes with a role in metabolism as well as defense response.
as an alternative to candidate gene approaches, pujolar et al.
<cit>  tested for footprints of selection in glass eels at the genome level using  <dig>  snps generated by rad sequencing.
a total of  <dig> potentially locally selected snps were identified.
candidate genes for local selection constituted a wide array of functions, including calcium signalling, neuroactive ligand-receptor interaction and circadian rhythm.
the power and efficiency of next generation sequencing  technologies are enabling research use of genomic data to address ecological and evolutionary questions at a genome-wide scale for model and non-model species  <cit> .
the insights that are obtained through ngs methods, as well as high throughput genotyping methods in general, have led to unprecedented progress in many areas, from bridging ecological and evolutionary concepts to identifying the molecular basis of local selection and adaptation .
the aim of our study is to test for selection acting upon different life cycle stages separated by metamorphosis in european eel, in particular between glass eels  and silver eels .
first, we used a candidate gene approach and genotyped individuals from three sampling locations  using a panel of  <dig> coding-gene snps.
second, we performed rad-sequencing of individuals from two sampling locations , which allowed us to test for signatures of local selection at the genome level using a total of  <dig>  snps widely distributed across the genome.
following the shifts in selection exerted by the dramatic physiological, morphological and ecological changes that accompany metamorphosis, our prediction is that different genes and gene pathways will be under selection at different life stages, and those will appear as outlier loci  when comparing glass eels vs. silver eels.
ultimately, the identification of genes showing marked differences when comparing life stages separated by metamorphosis can provide insights into how european eel can cope with the incredible variety of conditions and environments encountered throughout its complex life cycle.
it can also provide general insights into adaptive genomic evolution in natural populations of marine fishes.
RESULTS
snp genotyping
details of all polymorphic loci, including frequency of the most common allele across all sampling locations in our study in silver eels and glass eels are presented in table  <dig>  a total of  <dig> out of  <dig> snps were polymorphic, although at  <dig> loci minor allele frequency  was <  <dig>  in all samples.
genetic diversity values are summarized in table  <dig>  genetic diversity measures were very similar across samples and no significant differences were found across sampling locations in silver eels, i.e.
he ; ar .
moreover, no differences were found in genetic diversity measures when silver eels were compared to glass eels .
three loci deviated significantly from hwe at all locations  but were not excluded from the analysis, since departures from hwe might be due to selection.table  <dig> details of all polymorphic loci, including frequency of the most common allele across all silver eel  and glass eel  samples
genetic differentiation across silver eel samples was low and not significant .
a similar low genetic differentiation was found when considering all silver eel and glass eel samples  or when comparing pooled silver eels vs. pooled glass eels .
low fst values were also obtained when compared silver eels vs. glass eels in all samples: spain , ireland  or iceland .
prior to the selection analysis, we investigated the presence of hybrids in the silver eel data set using structure.
two individuals from iceland  were identified as hybrids showing a 50 % admixture proportion, and were consequently removed from the analysis .
results from outlier tests are summarized in table  <dig>  using lositan, a total of  <dig> outliers were identified when comparing all samples in our study : gapdh, ald_r and clic <dig>  when comparing the  <dig> silver eel samples, two outliers were identified: clic <dig> and ldhb.
when comparing silver eels vs. glass eels in all samples separately, different outlier loci were identified at each location: clic <dig> when comparing silver eels and glass eels from spain; cst and csde <dig> when comparing silver eels and glass eels from ireland; and gapdh when comparing silver eels and glass eels from iceland.
gapdh was also detected as outlier when comparing pooled silver eels vs. pooled glass eels.
when using bayescan, fewer outliers were identified in comparison with lositan, all showing high fst values: gapdh  and ald_r  when considering all samples and gapdh  when comparing silver eels and glass eels from iceland.table  <dig> candidate genes under selection at  <dig> snps in lositan and bayescan.
se = silver eels; ge = glass eels
rad sequencing
after sequencing of the rad libraries, average number of reads  per individual was  <dig>  million for silver eels and  <dig>  million for glass eels.
after trimming to 75 bp and quality filtered, the average number of high quality reads retained was  <dig>  million  for silver eels and  <dig>  million  for glass eels.
a similar percentage of reads were uniquely aligned to the european eel draft genome , while  <dig> - <dig>  % of sequences did not align and  <dig>  % were discarded due to multiple alignments.
aligned reads were then assembled into a total of  <dig>  loci using stacks .
after discarding  <dig>  % of loci due to insufficient coverage,  <dig>  loci were used to construct a catalog of loci of snps for all individuals.
at this point, a more strict filtering was applied and we eliminated  <dig> loci due to extremely high coverage ,  <dig> loci at which all individuals were either all heterozygotes or all homozygotes, and  <dig>  loci due to the presence of more than  <dig> alleles in a single individual, possibly reflecting paralogs or sequencing error.
after a final filtering step selecting only loci genotyped in > <dig>  % of individuals in all sampling locations, a total of  <dig>  rad loci were retained.
using populations in stacks, a total of  <dig>  snps were discovered, including  <dig>  snps with a minor allele frequency >  <dig> .table  <dig> statistics describing the distribution of different properties of rad sequences after each step of filtering , alignment to the eel draft genome  and assemblage into loci  in silver eels  and glass eels
measures of genetic diversity at  <dig>  loci at all sampling locations are summarized in table  <dig>  the valencia silver eel sample showed similar heterozygosities  and nucleotide diversity  compared to the valencia glass eel sample  and the burrishoole glass eel sample .
the burrishoole silver eel sample showed slightly lower diversity , presumably due to lower sample size.table  <dig> summary of genetic diversity indices at  <dig>  rad-loci including observed  and expected heterozygosities  and nucleotide diversity  considering only variant positions and considering all positions in all silver eel  and glass eel  samples
prior to the analysis of local selection, the presence of hybrids in the data set was assessed in structure using a subset of diagnostic snps between north atlantic eels and previously analyzed rad-sequenced american eels for comparison.
a scenario with k =  <dig> groups corresponding to the two north atlantic eel species was suggested.
within european eel, all rad-sequenced silver eels were identified as pure european eel, with no hybrids in the data set.
in the rad data set, outlier tests were first conducted comparing silver eels  and glass eels  from valencia using  <dig>  snps.
a total of  <dig> snps putatively under selection were identified by lositan and  <dig> snps by bayescan.
since the lositan outliers encompassed those outliers identified by bayescan, the rest of the analysis was conducted only for the lositan outliers.
out of the  <dig> candidate snps, a hit with a gene was obtained for  <dig>  of the snps, while the remaining  <dig> snps  were located in noncoding regions of the genome.
among hits,  <dig> were located in introns and  <dig> in exons, including  <dig> in complete coding sequences .
hits represented a total of  <dig> unique genes, of which  <dig>  genes were successfully annotated using blastx.
subsequently, the kegg pathway approach for higher-order functional annotation was implemented in david.
using zebrafish as reference genome, a total of  <dig> zebrafish genes homologous to european eel were mapped to kegg pathways.
enriched kegg pathways using a standard setting of gene count =  <dig> are summarized in table  <dig>  the pathways with the highest number of genes were mapk/erk signalling, calcium signalling, focal adhesion, cell adhesion and gnrh signalling.
a list of all annotated genes is provided in additional file 1: table s <dig> table  <dig> over-represented kegg pathways when comparing glass eels and silver eels from valencia , including gene count
we also tested for outliers between silver eels and glass eels from burrishoole, although adult sample size was low , as a way to confirm the results above.
a total of  <dig> putative snps under selection were identified by lositan and  <dig> snps by bayescan, the latter being all included in the list of lositan outliers.
among all outlier snps, a hit with a gene was obtained for  <dig> of the snps,  <dig> corresponding to introns and  <dig> to exons .
in total,  <dig>  of the hits were successfully annotated using blastx.
subsequently, a total of  <dig> zebrafish genes homologous to european eel were mapped to kegg pathways in david.
the pathway with the highest number of genes was endocytosis , while other highly-represented pathways included regulatory and signalling pathways .
importantly, despite the limited number of rad-sequenced adults from burrishoole, over-represented pathways were similar to the ones found when comparing silver eels and glass eels from valencia.
shared over-represented pathways included signalling  pathways and cell and focal adhesion.
a list of all annotated genes is provided in additional file 2: table s <dig> table  <dig> over-represented kegg pathways when comparing glass eels and silver eels from burrishole , including gene count
finally, average fst values between silver eels and glass eels from valencia calculated using a 50-kb sliding window were plotted for the  <dig> largest scaffolds.
fst was low throughout the scaffolds, with just a few narrow peaks.
no regions of the scaffolds with pronounced divergence peaks were observed, consistent with panmixia removing any effect of diversifying selection from each new generation.
similar results were obtained when using alternative  sliding windows.
figure  <dig> shows an example of the plots obtained for the three largest scaffolds  in the european eel draft genome.
a total of  <dig>   <dig> and  <dig> peaks representing outlier snps with higher fst relative to the background were observed in scaffolds  <dig>   <dig> and  <dig>  respectively.
the average distance between outlier snps was around  <dig>  bp in scaffolds  <dig> and  <dig> and around  <dig>  bp in scaffold  <dig>  the closest distance between outlier snps was 9000 bp in scaffold  <dig>   <dig>  bp in scaffold  <dig> but around  <dig>  bp in scaffold  <dig> fig.
<dig> plots of average fst calculated using a 50-kb sliding window for the three largest scaffolds  in the european eel genome
discussion
evidence for selection acting upon different life stages in european eel
we examined the patterns of genomic diversity across life cycle stages in european eel, using a combined approach of candidate coding-gene snps and a large-scale genomic analysis of  <dig>  snps generated by rad sequencing.
we compared two life cycle stages separated by metamorphosis, glass eels  and silver eels , and identified signatures of directional selection.
all available information indicates that eel mortality in nature is very high and only a small fraction of the glass eels entering european coasts reach the silver eel stage and migrate back to the sargasso sea  <cit> .
bonhommeau et al.
<cit>  estimated a glass eel survival rate of 10 %, while Åström and dekker  <cit>  estimated a natural mortality rate of m =  <dig>  per year and a fishery mortality rate of f =  <dig>  per year.
hence, the observation of outlier loci showing high genetic differentiation when comparing glass eels vs. silver eels is consistent with the action of natural selection acting upon eels.
fst-based outlier tests are based on the detection of loci that show significantly high differentiation with significance determined by simulations assuming specific population structure models  <cit>  and are being extensively used at present in studies aiming at detecting signatures of selection on a genome scale , although results should be interpreted with caution .
using the snp panel developed by gagnaire et al.
<cit>  in american eel, a total of four loci showed higher genetic differentiation than the background fst when comparing glass eel and silver eel samples.
gapdh  is a gene with a major metabolic function and catalyzes the conversion of glyceraldehyde 3-phosphate to d-glycerate  <dig> -biphosphate in the glycolysis pathway.
in this sense, gapdh has been linked to growth differences in gene expression studies in fishes  <cit> .
cst  is a gene involved in catalytic activity that takes part in defense response.
clic <dig>  is a gene involved in chloride ion transport, which is important for ph regulation, volume homeostasis, organic solute transport, cell migration, cell proliferation and differentiation.
csde <dig>  is a rna-binding protein involved in regulation of transcription.
however, all outliers were location-specific  and none was common across locations.
this suggests that if the outliers detected indeed do represent selection, then they do not result from a universal selection agent affecting eels in all places but rather location-specific factors affecting eels locally.
a much larger number of candidate genes putatively under selection were identified after screening a total of  <dig>  rad-generated snps across the genome:  <dig> when comparing glass and silver eels from spain and  <dig> in the case of ireland.
functional annotation analysis using david identified signal transduction pathways as the most over-represented.
signal transduction involves the binding of an extracellular signalling molecule  to a specific cell-surface receptor.
the activation of the receptor leads to an altered response inside the cell.
examples of cellular responses to extracellular stimulation that require signal transduction include changes in metabolism and gene expression.
following are some major pathways identified in our study:
- mapk/erk signalling: a complex key signalling pathway that is involved in the regulation of normal cell proliferation, survival, growth and differentiation  <cit> ; the pathway includes mitogen-activated protein kinases that ultimately activate transcription factors and alter gene transcription.
- gnrh signalling: secretion of gonadotropin-releasing hormone from the hypothalamus acts upon its receptor in the anterior pituitary to regulate the production and release of fsh  and lh ; together, fsh and lh regulate many aspects of gonadal function in both males and females, including normal growth, sexual development and reproductive function  <cit> .
- calcium signalling: calcium ions serve a number of important functions and regulate processes as diverse as fertilization, development, learning and memory, mitochondrial function, muscle contraction and secretion; calcium ions are also recognized as very important in ion exchange and osmoregulation  <cit> .
- insulin-like growth factor signalling: this pathway plays a central role in the neuroendocrine regulation of animal growth and development.
in fish, additional functions include osmoregulatory acclimation, reproductive development and tissue regeneration.
insulin-like growth factor signalling is mediated by two ligands, insulin-like growth factor  <dig>  and factor  <dig> , both of which were identified as outliers.
the production of igf- <dig> is stimulated by growth hormone and is positively correlated with growth as shown in coho salmon  <cit>  or sea bream  <cit> .
- focal adhesion: related to signalling pathways, focal adhesion was also over-represented in our analysis.
focal adhesions are large protein assembles through which both mechanical force and regulatory signals are transmitted and have central roles in cell migration and morphogenesis as well as regulating cell proliferation and differentiation  <cit> .
other over-represented pathways were related to metabolism  and detoxification of xenobiotics .
the latter included cytochrome cyp2j <dig>  a member of the cytochrome p <dig> superfamily of enzymes that catalyze many reactions involved in drug metabolism.
overall, when we consider the functions of the genes, it seems biologically plausible that the genes identified as outliers are indeed under selection.
many of the over-represented pathways were related to growth, while others could result from the different conditions and habitats that eels inhabit throughout their life cycle.
in this sense, examination of otolith data suggests a high plasticity of habitat use by eels  <cit> , with one or several movements between fresh and brackish waters throughout the lifetime of an individual.
when only a single habitat switch event was detected, it occurred between  <dig> and 5 years of age, which could explain the differences found in our study at osmoregulation genes between glass eels and silver eels.
when comparing across methods, a much larger number of genes putatively under selection were identified using the rad genome scan approach.
this is expected, since we screened only  <dig> snps with the american eel snp panel, while we interrogated over  <dig>  snps with the rad approach.
however, when considering percentage rather than total number of markers under selection, results were similar.
for instance, in the case of valencia we found  <dig>  % of snps putatively under selection using the rad approach , with a similar percentage  found using the american eel snp array.
in the case of burrishoole, candidate snps under selection were  <dig>  %  using the rad approach and  <dig>  %  using the american eel snp array.
finally, it should be noted that outliers were not shared across methods, which is explained by the different nature of the markers.
all snps included in the array were located in coding-genes, while the snps discovered using the rad approach were mostly located in non-coding genes, since rad tags are restriction site generated markers randomly distributed across the genome.
support for the adaptive decoupling hypothesis
the observation in our study of a large number of outlier loci showing higher fst values relative to the background when comparing glass eels and silver eels fits the prediction that in the case of animals with complex life cycles, different genes and gene pathways will be under selection at different life stages.
this is in accordance with the adaptive decoupling hypothesis for the benefits of metamorphosis  <cit> , which predicts no correlation between traits separated by metamorphosis, thereby each life stage can respond independently to its unique selective pressures.
moran  <cit>  hypothesized that the genetic decoupling of pre- and post-metamorphosis life stages explains the origin and persistence of complex life cycles, as alternative phases can be regarded as adaptations for a more effective exploitation of resources and adaptations to perform phase-specific tasks.
in the case of eels, the silvering metamorphosis, which represents the passage from juvenile to adult, is accompanied by drastic modifications at the physiological, morphological and ecological level that could explain the shifts in selection acting across life cycle stages.
changes occur both externally  and internally .
while the juvenile stage is perfectly adapted for feeding, adults are not able to feed anymore following metamorphosis, relying solely on fat reserves to reach the sargasso sea.
however, the adult stage is best adapted for migration, mating and reproducing.
if selection were complementary between life cycle stages separated by metamorphosis, the same genes and pathways would be expected to be under selection before and after metamorphosis and no outlier loci showing high differentiation would be expected.
by contrast, in our study we found up to  <dig> outlier loci, suggestive of opposing selection between life cycle stages, as predicted by the adaptive decoupling hypothesis.
alternatively, results in our study could reflect changes in genetic frequencies over time rather than a selection effect.
one possibility we cannot rule out is a temporal effect, since juveniles and adults in our sampling were not from the same cohort.
following the same cohort in time would be ideal to test our hypothesis of opposing selection during different life stages separated by metamorphosis, however this is virtually impossible in eels.
age at maturity  is highly variable in eels, ranging from  <dig> to 20 years or more  <cit> .
this means that individuals from the same cohort will become silver eels at different times and that adult eel samples for genetic studies are mostly made up of a mix of different cohorts.
nevertheless, the observation of a parallel pattern of genetic differentiation in valencia and burrishoole when comparing glass eels and silver eels, with many over-represented genes and pathways being shared by the two locations, lends support to a selection effect.
genomic distribution of candidate genes under selection
genomic regions displaying elevated differentiation relative to the rest of the genome  have been described in many species, as exemplified in the case of sticklebacks  <cit> .
in marine fishes, genomic islands of divergence might be unexpected because of their extremely high effective population sizes .
due to the effects of ne on the level of linkage disequilibrium, a fast decay of linkage disequilibrium should be generally expected , which in turn might preclude hitchhiking and ultimately the observation of genomic islands of divergence.
that might be the case of eels.
in our study comparing glass eels and silver eels, outlier snps showing high genetic differentiation consistent with selection did not group into clusters but were generally spread across the genome.
similarly, no apparent genomic islands of divergence were found when investigating the genomic distribution of outlier snps between european and american eel  <cit> .
in contrast to the pattern observed in european eel, genomic clustering of some highly divergent snps was observed in atlantic herring  <cit> , a species with a very large effective population size.
elevated genomic differentiation across large genomic blocks  was also reported in atlantic cod  <cit> , which suggests that genomic islands of divergence can occur in marine fishes.
however, it is likely that the different pattern observed in eels  vs. atlantic herring and atlantic cod  might be due to the different impact of evolutionary forces acting upon the panmictic european eel and other species that are genetically sub-structured.
therefore, unlike in eels, significant linkage disequilibrium might occur in atlantic herring and atlantic cod, thus allowing hitchhiking to accumulate, which ultimately results in the clustering of genes with localized elevated differentiation relative to the background.
finally, it should be noted that by using an fst-outlier approach, the number of loci under selection might be underestimated.
while standard tests of selection  are powerful tools to detect “hard selective sweeps”, in which a new advantageous mutation arises and spreads quickly to fixation due to natural selection  <cit> , other scenarios might be more difficult to detect.
those include soft sweeps, in which an allele already present in the population  becomes selectively favoured or when multiple independent mutations at a single locus are all favoured  <cit> , and polygenic adaptation, in which simultaneous selection occurs on variants at many loci  <cit> .
both scenarios lead to shifts in allele frequencies rather than fixation, thus tend to be more difficult to detect than hard sweeps using standard tests of selection  <cit> .
considering the high historical effective population size estimated for the european eel  and associated high genetic variability  <cit> , soft sweeps might be more common than hard sweeps and hence our study might have uncovered only a fraction of the genes under selection across life stages in european eel.
CONCLUSIONS
our data supports the adaptive decoupling hypothesis for the benefits of metamorphosis in european eel since genes and gene pathways under selection were different in pre- and post-silvering metamorphosis.
the differences found between juveniles and adults suggests that partitioning the life cycle into discrete stages may be more effective than a single stage in the case of eels.
this way,each life stage can perform specific tasks more effectively, i.e.
feeding in glass eels, reproducing in silver eels.

 many animals show complex life cycles organized into morphologically distinct phases separated by abrupt metamorphic transitions, as opposed to single static or continuously changing phases. complex life cycles are ubiquitous in nature and have evolved many times independently. life stages are believed to represent alternative adaptations for optimal food and niche exploitation as well as conflicting tasks. since darwin, evolutionary biologists have been interested in understanding the genetic associations between life cycle stages and to what extent discrete phases are free to evolve independently from one another. metamorphosis marks drastic morphological, physiological, behavioural and ecological changes in the life cycles of animals. given the dramatic changes associated with metamorphosis, selection could differ before and after metamorphosis, and opposing selection might be more common than complimentary selection. in regard to the benefits of metamorphosis, the adaptive decoupling hypothesis predicts that traits separated by metamorphosis should be genetically uncorrelated, allowing distinct phases to respond independently to different selective forces, without correlated negative effects on traits of alternative phases. studies testing the adaptive decoupling hypothesis have found contradictory results and the genetic associations between life cycle stages separated by metamorphosis remain poorly understood. our species of interest is the european eel, a facultative catadromous fish with a particularly complex life cycle that includes two metamorphoses. after spawning in the remote sargasso sea, larvae of european eel are transported to the coasts of europe and north africa following the gulf stream and north atlantic current. on reaching the continental shelf, eels undergo a first metamorphosis from larvae into glass eels, which complete their migration into continental feeding habitats as yellow eels. after an extensive feeding/growing period, eels undergo a second metamorphosis into silver eels. the so-called “silvering metamorphosis” encompasses morphological as well as physiological modifications, together with the development of gonads. these changes are beforehand preparation for the future spawning migration back to the sargasso sea, where eels reproduce and die. the european eel is an ideal species in which to study local selection. first, it presents a large effective population size that renders natural selection the major force determining genetic differences; hence the role of random genetic drift is expected to be negligible. second, it is present across extremely heterogenous environments in terms of temperature, salinity, substrate, depth and productivity along its geographic distribution. despite such a wide distribution range, there is recent conclusive evidence for panmixia in european eel, i.e. the existence of a single randomly mating population. in the most comprehensive study to date genotyping over individuals obtained throughout all the distribution range in europe at microsatellite loci, als et al. showed a very low and nonsignificant genetic diffferentiation across europe and a lack of substructuring among larvae collected in the sargasso sea. moreover, no significant genetic differentiation was observed when comparing the samples obtained in europe vs. the larvae obtained from the spawning area. panmixia was also confirmed at the genomic level in a study using a large dataset of >  snps from rad-sequenced european eels, which showed low levels of genetic differentiation. previous studies based on cohort analysis showed an unpatterned genetic heterogeneity as samples did not group together according to sampling location or cohort, and consequently no pattern on isolation-by-distance or isolation-by-time was detected. if european eel larvae showed phylopatry to the parental original freshwater habitats, genetic differences would be expected across europe; hence, the lack of genetic structuring found suggest no larval homing and random larval migratory routes. one consequence of panmixia and random dispersal of larvae across habitats is that long-term local adaptation is not possible in eels, despite the high potential for selective responses due to high mortalities in both early and late life stages. any signature of spatially varying selection in a given generation is expected to be lost in the subsequent generation, preventing heritable trans-generational local adaptation. however, single-generation signatures of local selection are still detectable. studies of adaptive evolution in european eel have focused on the detection of signatures of local selection in glass eels. using a panel of coding-gene single nucleotide polymorphisms, ulrik et al. found signatures of selection at loci in european eel, which constituted genes with a role in metabolism as well as defense response. as an alternative to candidate gene approaches, pujolar et al. tested for footprints of selection in glass eels at the genome level using snps generated by rad sequencing. a total of potentially locally selected snps were identified. candidate genes for local selection constituted a wide array of functions, including calcium signalling, neuroactive ligand-receptor interaction and circadian rhythm. the power and efficiency of next generation sequencing technologies are enabling research use of genomic data to address ecological and evolutionary questions at a genome-wide scale for model and non-model species. the insights that are obtained through ngs methods, as well as high throughput genotyping methods in general, have led to unprecedented progress in many areas, from bridging ecological and evolutionary concepts to identifying the molecular basis of local selection and adaptation. the aim of our study is to test for selection acting upon different life cycle stages separated by metamorphosis in european eel, in particular between glass eels and silver eels. first, we used a candidate gene approach and genotyped individuals from three sampling locations using a panel of coding-gene snps. second, we performed rad-sequencing of individuals from two sampling locations, which allowed us to test for signatures of local selection at the genome level using a total of snps widely distributed across the genome. following the shifts in selection exerted by the dramatic physiological, morphological and ecological changes that accompany metamorphosis, our prediction is that different genes and gene pathways will be under selection at different life stages, and those will appear as outlier loci when comparing glass eels vs. silver eels. ultimately, the identification of genes showing marked differences when comparing life stages separated by metamorphosis can provide insights into how european eel can cope with the incredible variety of conditions and environments encountered throughout its complex life cycle. it can also provide general insights into adaptive genomic evolution in natural populations of marine fishes.  snp genotyping details of all polymorphic loci, including frequency of the most common allele across all sampling locations in our study in silver eels and glass eels are presented in table  a total of out of snps were polymorphic, although at loci minor allele frequency was <  in all samples. genetic diversity values are summarized in table  genetic diversity measures were very similar across samples and no significant differences were found across sampling locations in silver eels, i.e. he; ar. moreover, no differences were found in genetic diversity measures when silver eels were compared to glass eels. three loci deviated significantly from hwe at all locations but were not excluded from the analysis, since departures from hwe might be due to selection.table details of all polymorphic loci, including frequency of the most common allele across all silver eel and glass eel samples genetic differentiation across silver eel samples was low and not significant. a similar low genetic differentiation was found when considering all silver eel and glass eel samples or when comparing pooled silver eels vs. pooled glass eels. low fst values were also obtained when compared silver eels vs. glass eels in all samples: spain, ireland or iceland. prior to the selection analysis, we investigated the presence of hybrids in the silver eel data set using structure. two individuals from iceland were identified as hybrids showing a 50 % admixture proportion, and were consequently removed from the analysis. results from outlier tests are summarized in table  using lositan, a total of outliers were identified when comparing all samples in our study: gapdh, ald_r and clic when comparing the silver eel samples, two outliers were identified: clic and ldhb. when comparing silver eels vs. glass eels in all samples separately, different outlier loci were identified at each location: clic when comparing silver eels and glass eels from spain; cst and csde when comparing silver eels and glass eels from ireland; and gapdh when comparing silver eels and glass eels from iceland. gapdh was also detected as outlier when comparing pooled silver eels vs. pooled glass eels. when using bayescan, fewer outliers were identified in comparison with lositan, all showing high fst values: gapdh and ald_r when considering all samples and gapdh when comparing silver eels and glass eels from iceland.table candidate genes under selection at snps in lositan and bayescan. se = silver eels; ge = glass eels rad sequencing after sequencing of the rad libraries, average number of reads per individual was million for silver eels and million for glass eels. after trimming to 75 bp and quality filtered, the average number of high quality reads retained was million for silver eels and million for glass eels. a similar percentage of reads were uniquely aligned to the european eel draft genome, while -  % of sequences did not align and  % were discarded due to multiple alignments. aligned reads were then assembled into a total of loci using stacks. after discarding  % of loci due to insufficient coverage, loci were used to construct a catalog of loci of snps for all individuals. at this point, a more strict filtering was applied and we eliminated loci due to extremely high coverage, loci at which all individuals were either all heterozygotes or all homozygotes, and loci due to the presence of more than alleles in a single individual, possibly reflecting paralogs or sequencing error. after a final filtering step selecting only loci genotyped in >  % of individuals in all sampling locations, a total of rad loci were retained. using populations in stacks, a total of snps were discovered, including snps with a minor allele frequency > .table statistics describing the distribution of different properties of rad sequences after each step of filtering, alignment to the eel draft genome and assemblage into loci in silver eels and glass eels measures of genetic diversity at loci at all sampling locations are summarized in table  the valencia silver eel sample showed similar heterozygosities and nucleotide diversity compared to the valencia glass eel sample and the burrishoole glass eel sample. the burrishoole silver eel sample showed slightly lower diversity, presumably due to lower sample size.table summary of genetic diversity indices at rad-loci including observed and expected heterozygosities and nucleotide diversity considering only variant positions and considering all positions in all silver eel and glass eel samples prior to the analysis of local selection, the presence of hybrids in the data set was assessed in structure using a subset of diagnostic snps between north atlantic eels and previously analyzed rad-sequenced american eels for comparison. a scenario with k =  groups corresponding to the two north atlantic eel species was suggested. within european eel, all rad-sequenced silver eels were identified as pure european eel, with no hybrids in the data set. in the rad data set, outlier tests were first conducted comparing silver eels and glass eels from valencia using snps. a total of snps putatively under selection were identified by lositan and snps by bayescan. since the lositan outliers encompassed those outliers identified by bayescan, the rest of the analysis was conducted only for the lositan outliers. out of the candidate snps, a hit with a gene was obtained for of the snps, while the remaining snps were located in noncoding regions of the genome. among hits, were located in introns and in exons, including in complete coding sequences. hits represented a total of unique genes, of which genes were successfully annotated using blastx. subsequently, the kegg pathway approach for higher-order functional annotation was implemented in david. using zebrafish as reference genome, a total of zebrafish genes homologous to european eel were mapped to kegg pathways. enriched kegg pathways using a standard setting of gene count =  are summarized in table  the pathways with the highest number of genes were mapk/erk signalling, calcium signalling, focal adhesion, cell adhesion and gnrh signalling. a list of all annotated genes is provided in additional file 1: table s table over-represented kegg pathways when comparing glass eels and silver eels from valencia, including gene count we also tested for outliers between silver eels and glass eels from burrishoole, although adult sample size was low, as a way to confirm the results above. a total of putative snps under selection were identified by lositan and snps by bayescan, the latter being all included in the list of lositan outliers. among all outlier snps, a hit with a gene was obtained for of the snps, corresponding to introns and to exons. in total, of the hits were successfully annotated using blastx. subsequently, a total of zebrafish genes homologous to european eel were mapped to kegg pathways in david. the pathway with the highest number of genes was endocytosis, while other highly-represented pathways included regulatory and signalling pathways. importantly, despite the limited number of rad-sequenced adults from burrishoole, over-represented pathways were similar to the ones found when comparing silver eels and glass eels from valencia. shared over-represented pathways included signalling pathways and cell and focal adhesion. a list of all annotated genes is provided in additional file 2: table s table over-represented kegg pathways when comparing glass eels and silver eels from burrishole, including gene count finally, average fst values between silver eels and glass eels from valencia calculated using a 50-kb sliding window were plotted for the largest scaffolds. fst was low throughout the scaffolds, with just a few narrow peaks. no regions of the scaffolds with pronounced divergence peaks were observed, consistent with panmixia removing any effect of diversifying selection from each new generation. similar results were obtained when using alternative sliding windows. figure  shows an example of the plots obtained for the three largest scaffolds in the european eel draft genome. a total of and peaks representing outlier snps with higher fst relative to the background were observed in scaffolds and respectively. the average distance between outlier snps was around  bp in scaffolds and and around  bp in scaffold the closest distance between outlier snps was 9000 bp in scaffold  bp in scaffold but around  bp in scaffold fig. plots of average fst calculated using a 50-kb sliding window for the three largest scaffolds in the european eel genome discussion evidence for selection acting upon different life stages in european eel we examined the patterns of genomic diversity across life cycle stages in european eel, using a combined approach of candidate coding-gene snps and a large-scale genomic analysis of snps generated by rad sequencing. we compared two life cycle stages separated by metamorphosis, glass eels and silver eels, and identified signatures of directional selection. all available information indicates that eel mortality in nature is very high and only a small fraction of the glass eels entering european coasts reach the silver eel stage and migrate back to the sargasso sea. bonhommeau et al. estimated a glass eel survival rate of 10 %, while Åström and dekker estimated a natural mortality rate of m =  per year and a fishery mortality rate of f =  per year. hence, the observation of outlier loci showing high genetic differentiation when comparing glass eels vs. silver eels is consistent with the action of natural selection acting upon eels. fst-based outlier tests are based on the detection of loci that show significantly high differentiation with significance determined by simulations assuming specific population structure models and are being extensively used at present in studies aiming at detecting signatures of selection on a genome scale, although results should be interpreted with caution. using the snp panel developed by gagnaire et al. in american eel, a total of four loci showed higher genetic differentiation than the background fst when comparing glass eel and silver eel samples. gapdh is a gene with a major metabolic function and catalyzes the conversion of glyceraldehyde 3-phosphate to d-glycerate -biphosphate in the glycolysis pathway. in this sense, gapdh has been linked to growth differences in gene expression studies in fishes. cst is a gene involved in catalytic activity that takes part in defense response. clic is a gene involved in chloride ion transport, which is important for ph regulation, volume homeostasis, organic solute transport, cell migration, cell proliferation and differentiation. csde is a rna-binding protein involved in regulation of transcription. however, all outliers were location-specific and none was common across locations. this suggests that if the outliers detected indeed do represent selection, then they do not result from a universal selection agent affecting eels in all places but rather location-specific factors affecting eels locally. a much larger number of candidate genes putatively under selection were identified after screening a total of rad-generated snps across the genome: when comparing glass and silver eels from spain and in the case of ireland. functional annotation analysis using david identified signal transduction pathways as the most over-represented. signal transduction involves the binding of an extracellular signalling molecule to a specific cell-surface receptor. the activation of the receptor leads to an altered response inside the cell. examples of cellular responses to extracellular stimulation that require signal transduction include changes in metabolism and gene expression. following are some major pathways identified in our study: - mapk/erk signalling: a complex key signalling pathway that is involved in the regulation of normal cell proliferation, survival, growth and differentiation; the pathway includes mitogen-activated protein kinases that ultimately activate transcription factors and alter gene transcription. - gnrh signalling: secretion of gonadotropin-releasing hormone from the hypothalamus acts upon its receptor in the anterior pituitary to regulate the production and release of fsh and lh; together, fsh and lh regulate many aspects of gonadal function in both males and females, including normal growth, sexual development and reproductive function. - calcium signalling: calcium ions serve a number of important functions and regulate processes as diverse as fertilization, development, learning and memory, mitochondrial function, muscle contraction and secretion; calcium ions are also recognized as very important in ion exchange and osmoregulation. - insulin-like growth factor signalling: this pathway plays a central role in the neuroendocrine regulation of animal growth and development. in fish, additional functions include osmoregulatory acclimation, reproductive development and tissue regeneration. insulin-like growth factor signalling is mediated by two ligands, insulin-like growth factor and factor, both of which were identified as outliers. the production of igf- is stimulated by growth hormone and is positively correlated with growth as shown in coho salmon or sea bream. - focal adhesion: related to signalling pathways, focal adhesion was also over-represented in our analysis. focal adhesions are large protein assembles through which both mechanical force and regulatory signals are transmitted and have central roles in cell migration and morphogenesis as well as regulating cell proliferation and differentiation. other over-represented pathways were related to metabolism and detoxification of xenobiotics. the latter included cytochrome cyp2j a member of the cytochrome p superfamily of enzymes that catalyze many reactions involved in drug metabolism. overall, when we consider the functions of the genes, it seems biologically plausible that the genes identified as outliers are indeed under selection. many of the over-represented pathways were related to growth, while others could result from the different conditions and habitats that eels inhabit throughout their life cycle. in this sense, examination of otolith data suggests a high plasticity of habitat use by eels, with one or several movements between fresh and brackish waters throughout the lifetime of an individual. when only a single habitat switch event was detected, it occurred between and 5 years of age, which could explain the differences found in our study at osmoregulation genes between glass eels and silver eels. when comparing across methods, a much larger number of genes putatively under selection were identified using the rad genome scan approach. this is expected, since we screened only snps with the american eel snp panel, while we interrogated over snps with the rad approach. however, when considering percentage rather than total number of markers under selection, results were similar. for instance, in the case of valencia we found  % of snps putatively under selection using the rad approach, with a similar percentage found using the american eel snp array. in the case of burrishoole, candidate snps under selection were  % using the rad approach and  % using the american eel snp array. finally, it should be noted that outliers were not shared across methods, which is explained by the different nature of the markers. all snps included in the array were located in coding-genes, while the snps discovered using the rad approach were mostly located in non-coding genes, since rad tags are restriction site generated markers randomly distributed across the genome. support for the adaptive decoupling hypothesis the observation in our study of a large number of outlier loci showing higher fst values relative to the background when comparing glass eels and silver eels fits the prediction that in the case of animals with complex life cycles, different genes and gene pathways will be under selection at different life stages. this is in accordance with the adaptive decoupling hypothesis for the benefits of metamorphosis, which predicts no correlation between traits separated by metamorphosis, thereby each life stage can respond independently to its unique selective pressures. moran hypothesized that the genetic decoupling of pre- and post-metamorphosis life stages explains the origin and persistence of complex life cycles, as alternative phases can be regarded as adaptations for a more effective exploitation of resources and adaptations to perform phase-specific tasks. in the case of eels, the silvering metamorphosis, which represents the passage from juvenile to adult, is accompanied by drastic modifications at the physiological, morphological and ecological level that could explain the shifts in selection acting across life cycle stages. changes occur both externally and internally. while the juvenile stage is perfectly adapted for feeding, adults are not able to feed anymore following metamorphosis, relying solely on fat reserves to reach the sargasso sea. however, the adult stage is best adapted for migration, mating and reproducing. if selection were complementary between life cycle stages separated by metamorphosis, the same genes and pathways would be expected to be under selection before and after metamorphosis and no outlier loci showing high differentiation would be expected. by contrast, in our study we found up to outlier loci, suggestive of opposing selection between life cycle stages, as predicted by the adaptive decoupling hypothesis. alternatively, results in our study could reflect changes in genetic frequencies over time rather than a selection effect. one possibility we cannot rule out is a temporal effect, since juveniles and adults in our sampling were not from the same cohort. following the same cohort in time would be ideal to test our hypothesis of opposing selection during different life stages separated by metamorphosis, however this is virtually impossible in eels. age at maturity is highly variable in eels, ranging from to 20 years or more. this means that individuals from the same cohort will become silver eels at different times and that adult eel samples for genetic studies are mostly made up of a mix of different cohorts. nevertheless, the observation of a parallel pattern of genetic differentiation in valencia and burrishoole when comparing glass eels and silver eels, with many over-represented genes and pathways being shared by the two locations, lends support to a selection effect. genomic distribution of candidate genes under selection genomic regions displaying elevated differentiation relative to the rest of the genome have been described in many species, as exemplified in the case of sticklebacks. in marine fishes, genomic islands of divergence might be unexpected because of their extremely high effective population sizes. due to the effects of ne on the level of linkage disequilibrium, a fast decay of linkage disequilibrium should be generally expected, which in turn might preclude hitchhiking and ultimately the observation of genomic islands of divergence. that might be the case of eels. in our study comparing glass eels and silver eels, outlier snps showing high genetic differentiation consistent with selection did not group into clusters but were generally spread across the genome. similarly, no apparent genomic islands of divergence were found when investigating the genomic distribution of outlier snps between european and american eel. in contrast to the pattern observed in european eel, genomic clustering of some highly divergent snps was observed in atlantic herring, a species with a very large effective population size. elevated genomic differentiation across large genomic blocks was also reported in atlantic cod, which suggests that genomic islands of divergence can occur in marine fishes. however, it is likely that the different pattern observed in eels vs. atlantic herring and atlantic cod might be due to the different impact of evolutionary forces acting upon the panmictic european eel and other species that are genetically sub-structured. therefore, unlike in eels, significant linkage disequilibrium might occur in atlantic herring and atlantic cod, thus allowing hitchhiking to accumulate, which ultimately results in the clustering of genes with localized elevated differentiation relative to the background. finally, it should be noted that by using an fst-outlier approach, the number of loci under selection might be underestimated. while standard tests of selection are powerful tools to detect “hard selective sweeps”, in which a new advantageous mutation arises and spreads quickly to fixation due to natural selection, other scenarios might be more difficult to detect. those include soft sweeps, in which an allele already present in the population becomes selectively favoured or when multiple independent mutations at a single locus are all favoured, and polygenic adaptation, in which simultaneous selection occurs on variants at many loci. both scenarios lead to shifts in allele frequencies rather than fixation, thus tend to be more difficult to detect than hard sweeps using standard tests of selection. considering the high historical effective population size estimated for the european eel and associated high genetic variability, soft sweeps might be more common than hard sweeps and hence our study might have uncovered only a fraction of the genes under selection across life stages in european eel.  our data supports the adaptive decoupling hypothesis for the benefits of metamorphosis in european eel since genes and gene pathways under selection were different in pre- and post-silvering metamorphosis. the differences found between juveniles and adults suggests that partitioning the life cycle into discrete stages may be more effective than a single stage in the case of eels. this way,each life stage can perform specific tasks more effectively, i.e. feeding in glass eels, reproducing in silver eels. 
species showing complex life cycles provide excellent opportunities to study the genetic associations between life cycle stages, as selective pressures may differ before and after metamorphosis. the european eel presents a complex life cycle with two metamorphoses, a first metamorphosis from larvae into glass eels and a second metamorphosis into silver eels. adaptative decoupling hypothesiscomplex life cyclesmetamorphosisrad sequencingselectionissue-copyright-statement© the author 2015 we tested the hypothesis that different genes and gene pathways will be under selection at different life stages when comparing the genetic associations between glass eels and silver eels. many of the over-represented pathways were related to growth, while others could result from the different conditions that eels inhabit during their life cycle. functional annotation analysis identified signal transduction pathways as the most over-represented group of genes, including mapk/erk signalling, calcium signalling and gnrh signalling. using the rad approach, outlier tests identified a total of potentially selected snps. the observation of different genes and gene pathways under selection when comparing glass eels vs. silver eels supports the adaptive decoupling hypothesis for the benefits of metamorphosis. partitioning the life cycle into discrete morphological phases may be overall beneficial since it allows the different life stages to respond independently to their unique selection pressures. we used two sets of markers to test for selection: first, we genotyped individuals using a panel of coding-gene single nucleotide polymorphisms developed in american eel; second, we investigated selection at the genome level using a total of rad-sequencing generated snps widely distributed across the genome. this might translate into a more effective use of food and niche resources and/or performance of phase-specific tasks.
 species showing complex life cycles provide excellent opportunities to study the genetic associations between life cycle stages, as selective pressures may differ before and after metamorphosis. the european eel presents a complex life cycle with two metamorphoses, a first metamorphosis from larvae into glass eels and a second metamorphosis into silver eels. we tested the hypothesis that different genes and gene pathways will be under selection at different life stages when comparing the genetic associations between glass eels and silver eels.  we used two sets of markers to test for selection: first, we genotyped individuals using a panel of coding-gene single nucleotide polymorphisms developed in american eel; second, we investigated selection at the genome level using a total of rad-sequencing generated snps widely distributed across the genome. using the rad approach, outlier tests identified a total of potentially selected snps. functional annotation analysis identified signal transduction pathways as the most over-represented group of genes, including mapk/erk signalling, calcium signalling and gnrh signalling. many of the over-represented pathways were related to growth, while others could result from the different conditions that eels inhabit during their life cycle.  the observation of different genes and gene pathways under selection when comparing glass eels vs. silver eels supports the adaptive decoupling hypothesis for the benefits of metamorphosis. partitioning the life cycle into discrete morphological phases may be overall beneficial since it allows the different life stages to respond independently to their unique selection pressures. this might translate into a more effective use of food and niche resources and/or performance of phase-specific tasks. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords adaptative decoupling hypothesiscomplex life cyclesmetamorphosisrad sequencingselectionissue-copyright-statement© the author 2015 