BACKGROUND
fish in temperate lakes that have been formed since the last glaciation  provide good model systems for studying adaptive radiation and ecological speciation  <cit> .
according to the ecological theory of adaptive radiation, environmental gradients and intense competition for scarce resources lead to divergent natural selection that, in turn, produces phenotypic differentiation  <cit> .
reproductive isolation is thought to evolve as a by-product of divergent natural selection because traits responsible for reproductive isolation are usually adaptive  <cit> .
sympatric populations of north temperate freshwater fish with significant genetic differentiation and strong ecological divergence are well known in sticklebacks   <cit> , rainbow smelt )  <cit> , arctic char )  <cit>  or sockeye salmon )  <cit> .
but no group of fishes is as well known for adaptive radiations and complex speciation patterns in postglacial lakes as coregonid fishes   <cit> .
a recent review  <cit>  identified six potential modes of speciation in coregonus: sympatric speciation from a single founder lineage, repeated invasions from a single lineage, hybrid swarm radiation, and three variants of allopatric speciation; complete or following secondary contact with either subsequent genomic reinforcement or with subsequent ecological reinforcement, depending on the levels of reproductive isolation between the taxa  <cit> .
in order to distinguish from among these potential modes of speciation, population genetic information is required in addition to phylogenetic, biogeographical, morphological and ecological data.
specifically, lower genetic differentiation between sympatric than between allopatric populations provides evidence for hybrid swarm and sympatric speciation scenarios.
conversely, more pronounced reproductive isolation between sympatric populations than between allopatric populations provides evidence for allopatric speciation with reinforcement  <cit> .
ecological diversification leading to speciation has been repeatedly suggested in coregonus ; however, recent reviews  <cit>  have challenged the assumption that all cases of ecological segregation between sympatric populations provide valid evidence for ecological speciation.
nevertheless, particularly strong support for sympatric ecological speciation can be obtained from cases of parallel ecological divergence and genetic differentiation in similar systems  <cit> .
further evidence for ecological speciation can be gained from genome scans that detect the same loci under selection in different systems with similar environments  <cit> .
in a geographical context, the genetic distances among populations of the founder lineage of sympatric species should fit to an isolation-by-distance  model  <cit> , whereas adaptive divergence between the sympatric populations may disrupt the ibd patterns  <cit> .
subsequently, sympatric populations would be genetically different but with a geographical distance of zero, thereby reducing our ability to detect significant ibd.
within the coregonidae, the postglacial evolution of whitefish  and c. lavaretus  complexes) and the north america ciscoes  are comparatively well studied  <cit> .
several speciation modes have been suggested for these groups, but evidence for parallel sympatric speciation from a single founder lineage is weak in any of the systems  <cit> .
an alternative system to study putative parallel ecological speciation can be found within populations of the baltic ciscoes  complex), a group that is widespread in northern europe  <cit> .
sympatric populations of the c. albula complex occur in scandinavia and russia  <cit>  and have been reported from two deep, stratified clear-water lakes  <cit>  situated geographically close to one another in northern germany .
in lake stechlin , the autumn-spawning c. albula coexists with the smaller, spring-spawning c. fontanae  <cit> .
both species are pelagic zooplanktivores  <cit> , but differ substantially with respect to temperature-dependent metabolic adaptations  <cit> .
analysis of six microsatellite loci revealed very low levels of genetic differentiation between the sympatric populations in lake stechlin  <cit> .
in the nearby lake breiter luzin , two populations also coexist   <cit> , between which the microsatellite differentiation was also very low  <cit> .
while these results suggest a sympatric origin of each spring spawner, shared mtdna haplotypes revealed lower genetic relatedness between sympatric taxa  <cit> .
an important advance to our understanding of the origin of the sympatric pairs could come from a genetic comparison with other allopatric populations from the same geographical area.
furthermore, the few neutral microsatellite markers employed did not allow for the study of potentially parallel adaptive divergence in both lakes.
accordingly, the specific speciation mode of the sympatric populations has yet to be unambiguously determined.
here, we studied genetic differentiation within the c. albula complex from eight lakes using amplified fragment length polymorphism .
six lakes had single, autumn-spawning c. albula populations, whereas two lakes contained c. albula together with a sympatric species  .
we first examined the overall genetic structure of the  <dig> coregonus populations, with particular emphasis on comparing differentiation between allopatric and sympatric populations.
under the scenario of sympatric speciation, we expected to find greater genetic similarity between the sympatric populations within lakes than among lakes  <cit> .
species that have evolved recently may not have developed full genetic incompatibilities and thus may hybridize with little intrinsic fitness loss, therefore we also tested whether ongoing hybridization between sympatric populations may blur historical differentiation  <cit> .
second, we fit genetic distances to a stepwise ibd model and hypothesized a disruption of ibd if sympatric populations are included  <cit> .
third, we calculated the probability of hybridization between sympatric populations and fish from putative stocking source populations.
enhancement stocking in the commercially important coregonus lake populations is a common management practice  <cit> , and our earlier work has quantified how this can modify the genetic identity of the target populations and disrupt ibd  <cit> .
we hypothesized that hybridization with c. albula stocked from allopatric autumn-spawning populations would be more likely for the autumn-spawning c. albula from both lakes than for the sympatric spring-spawning c. fontanae or c. lucinensis.
finally, we tested for the presence of outlier loci that were shared in both sympatric pairs to find genetic evidence for parallel adaptive divergence  <cit> .
RESULTS
all coregonus populations and all aflp loci
using a multiplex aflp method  we scored  <dig> loci from  <dig> individuals, of which  <dig>  were polymorphic over the  <dig> populations.
differentiation  between the repeated samplings of populations in lake stechlin were not significantly different from zero , thus samples were pooled for each population in subsequent analyses.
the percentage of polymorphic loci varied from  <dig> - 92% within the  <dig> populations .
estimated with hs, the expected heterozygosity ranged from  <dig>  to  <dig> , with the highest values being found in the two c. albula populations that occur with the sympatric species .
lake names, geographical position and genetic diversity of the  <dig> coregonus albula complex populations sampled.
allopatric populations consist exclusively of c. albula.
sympatric populations are named by lake origin and the lake-specific species names.
n = number of individuals analysed, % polymorphic loci refers to the total of  <dig> loci analysed, hs = gene diversity with credibility intervals calculated by a bayesian approach as implemented in hickory, hs = the mean within-population expected heterozygosity .
there was significant genetic differentiation among the  <dig> populations, with  <dig> % of variation found among populations .
global values of θ  and θb  were nearly identical and also showed significant population structure.
all pairwise θ-values were significantly different from zero  and ranged from  <dig>   to  <dig>   .
all nodes of the nj tree of nei's genetic distances were supported by bootstrap values of at least 95% .
the tree grouped four lakes with single populations  as distinct from the two lakes with sympatric population pairs  .
lakes arend and behler appeared more closely related to stechlin and breiter luzin.
interestingly, the allopatric spring-spawners, c. fontanae and c. lucinensis, grouped more closely to one another than either did to its sympatric c. albula population .
pairwise genetic and geographical distances were not correlated for the entire dataset , providing evidence that an ibd model did not appropriately describe the pattern of genetic differentiation among all  <dig> populations .
an ibd model was also not appropriate to describe the genetic differentiation between all eight c. albula populations including those from lakes stechlin and breiter luzin .
in contrast, when only the six lakes with only a c. albula population were included, genetic distances increased significantly with geographical distance as predicted by an ibd model .
however, the pairwise difference between the closely located lakes ploener and behler  appeared to be slightly higher  than predicted by strict linear pattern .
sympatric pairs
lake stechlin
lake stechlin
genetic differentiation  for the two sympatric population pairs of the coregonus albula complex.
values were calculated with hickory based on all  <dig> aflp loci  and after removal of n =  <dig> loci putatively under selection at p =  <dig>  .
the analyses of potential gene flow within the two lakes by structure found some evidence for the existence of hybrids between sympatric populations .
the mean proportion  of any individual's genome that originated from its own population  <cit>  was generally ≥ 87% .
three individuals  showed elevated probabilities of misclassification  or hybridization in the f1-generation .
for  <dig> individuals, there was a high probability of f2- or older hybridizations with the sympatric population.
in contrast, the probability of f1-hybrids formed with stocked fish from lakes arend and tollense was zero for all populations, and only six individuals showed a high probability of f2- and older hybrids with stocked fish .
outlier loci
the explorative genome scans revealed very few loci to be positively exceeding neutral expectations  in the two lakes with sympatric population pairs.
more loci under putative selection were found in lake stechlin  than in lake breiter luzin .
no single locus was non-neutral in both lakes.
at the p =  <dig>  quantile,  <dig> % of loci  were positively non-neutral in lake stechlin, and  <dig> %  in breiter luzin.
at this quantile, three loci  were shared from both lakes .
after removal of all  <dig> loci putatively under selection above the p =  <dig>  quantile in either lake stechlin or breiter luzin, genetic differentiation based on the remaining loci declined considerably, in particular between the sympatric pairs .
θb-values were almost identical between c. albula and c. fontanae in lake stechlin  and c. albula and c. lucinensis in lake breiter luzin , and did not differ from the differentiation between c. fontanae and c. lucinensis  .
only differentiation between both c. albula populations was still lower , with its credibility interval not overlapping with those of any other pairwise comparisons .
genetic distances between the eight c. albula populations remained almost unaffected by the removal of outlier loci.
the nj-tree calculated from the putatively neutral aflp loci had an identical topology to the nj-tree based on all aflp loci , with only minimal changes in branch lengths .
discussion
conflicting evidence for sympatric speciation
our results suggest that the post-glacial evolution of sympatric coregonus pairs studied here is more complex than previously thought.
neither spring spawner occurred elsewhere in allopatry, supporting the hypothesis that they evolved by sympatric speciation from the autumn-spawning c. albula within each lake, as suggested previously  <cit> .
nonetheless, the pattern of genetic differentiation that we observed contradicts this hypothesis.
in cases of sympatric speciation, sympatric taxa should be more closely related  <cit> , but here the autumn-spawning c. albula were more similar to each other than either was to its sympatric spring spawner.
this was evident from the tests of pairwise population differentiation as well as from the nj-tree based on genetic distance.
these results were similar to an allozyme study of  <dig> finnish lakes, where allopatric autumn spawners formed a separate group to winter- or spring spawners  <cit> .
the complexity of the evolutionary history is evidenced by the somewhat conflicting results of previous genetic analyses.
six microsatellite loci revealed greater genetic similarity between sympatric spring- and autumn-spawners within lakes, thus suggesting ongoing gene flow and a sympatric origin  <cit> .
in contrast, mtdna from the same study  <cit>  qualitatively support the aflp results here, that differentiation between sympatric taxa is greater than between allopatric taxa.
in particular, the two most abundant of four recorded haplotypes were shared by c. albula in both lakes.
one of these was shared with the stechlin sympatric c. fontanae and the other was shared with the lake breiter luzin sympatric c. lucinensis.
c. fontanae and c. lucinensis shared no haplotypes  <cit> .
the reason for the discrepancy between the microsatellite and aflp data in one of the population pairs is not clear.
the large number of aflp loci employed here  should provide a better estimate of divergence than microsatellite loci , although we found evidence that not all loci were strictly neutral.
the two marker systems produced similar estimates of genetic structure for populations of brown trout  in  <dig> norwegian lakes, although admixture was greater based on aflp  <cit> .
aflp results were more congruent than mtdna with morphological characters in mormyrid electric fishes, likely due to mitochondrial introgression among lineages  <cit> .
accordingly, aflp seem to be an appropriate tool to examine the genetic differentiation of incipient ecological speciation  <cit> .
but as noted above, our results suggest that not all aflp markers were strictly neutral.
removing the loci putatively under selection reduced the within-lake genetic distances considerably, and differentiation based on the remaining neutral aflp loci was more similar to the pattern obtained from the neutral microsatellite data.
interestingly, the genetic distance to other, allopatric c. albula populations remained largely unaffected.
previous studies similarly found that differentiation between ecologically or morphologically distinct populations can be maintained for a small portion of the genome, whereas gene exchange continues to prevent divergence at the majority of loci  <cit> .
only three outlier loci occurred in both sympatric pairs at p >  <dig> .
it is possible that such a small number of loci positively exceeding neutral expectations might have occurred just by chance, thus providing little evidence in support of the hypothesis that the sympatric pairs result from parallel sympatric speciation  <cit> .
alternatively, it may be that only a few genes are involved.
the few shared loci deserve further attention since recent analyses in the c. clupeaformis complex in north america indicated that these θ outliers can be significantly clustered around quantitative trait loci  involved in ecologically based reproductive isolation  <cit> .
proportions of outlier loci substantially exceeding 1% were found in other studies applying a similar pairwise approach  <cit> .
the presumed modes of ecological segregation have been well studied in lake stechlin, where sympatric taxa  perform diel vertical migrations with different amplitudes  <cit>  and they differ in their final temperature preferendum and their metabolic costs of swimming  <cit> .
the commonly observed benthic-limnetic habitat and diet segregations typical for whitefish do not occur in stechlin  <cit> .
such a pattern of physiological segregation but low trophic differentiation presumably exists for the species pair in lake breiter luzin, since both lakes are deep, with steep temperature gradients and overall low productivity  <cit> .
recent data suggest c. albula and c. lucinensis in breiter luzin have similar diets  <cit> , as in lake stechlin  <cit> .
however, adaptive divergence may be weaker in lake breiter luzin than in lake stechlin.
for example, c. lucinensis and c. albula do not show the same strong vertical microhabitat segregation  as found between c. fontanae and c. albula in lake stechlin  <cit> .
a weaker adaptive divergence is supported by the lower genetic distance and a substantially lower number of loci putatively under selection in lake breiter luzin than in lake stechlin.
accordingly, these species pairs would resemble 'isolation by adaptation' , a process in which levels of genetic differentiation among population pairs will be positively correlated with degree of adaptive phenotypic divergence  <cit> .
stocking and hybridization
an important part of our analysis was to determine whether the genetic similarity of stechlin and breiter luzin c. albula was a result of modification by stocking and hybridization.
enhancement stocking of vendace populations by freshly hatched larvae  obtained primarily from two source populations  has been practised in germany for at least  <dig> years, with the aim of stabilizing natural recruitment and fisheries yield of sympatric and allopatric c. albula populations  <cit> .
because both putative stocking sources contain only autumn-spawning c. albula populations, hybridization with c. albula in stechlin and breiter luzin was hypothesized to be more likely than hybridization with the spring-spawning c. fontanae and c. lucinensis.
our results support this hypothesis.
furthermore, our recent analysis of the genetic effects of stocking on  <dig> allopatric c. albula populations revealed a reduction of genetic distance by about 10% between lakes stocked intensively with a genetically similar source  <cit> .
accordingly, a weak secondary homogenization between lakes stechlin and breiter luzin by stocking from lakes arend or tollense cannot be ruled out, but the evidence is not strong enough to conclude that the similarity of the autumn spawners is an exclusive effect of current fisheries management practices.
hybridization within the lakes could also contribute to the observed patterns, whereby introgression of c. albula genetic polymorphisms may obscure any historical similarities of the two spring spawners.
because species that have evolved since the most recent glaciation about  <dig>  years ago may not have developed full genetic incompatibilities, they may hybridize with little fitness loss  <cit> .
the probability that single individuals have parents or grandparents from the other population was low, but greater than zero in all four populations.
therefore, gene flow between the sympatric populations may not be completely interrupted, despite the differing spawning times.
weak ongoing hybridization between the sympatric populations can also be inferred from the higher heterozygosity of sympatric compared with allopatric c. albula populations  <cit> .
in this analysis, we used a high prior probability of pure ancestry similarly applied to the analysis of sympatric whitefish populations from the canadian cliff lake  <cit> .
this sympatric whitefish population pair was identified as being reproductively isolated by aflp-markers   <cit> , but nevertheless encompassed seven out of  <dig> individuals with strong evidence for mixed ancestry  <cit> .
accordingly, occasional hybridization within the two sympatric populations of the c. albula complex studied here cannot be excluded.
large-scale genetic structure of coregonus
an important finding was that the sympatric populations of lakes stechlin and breiter luzin were genetically distinct from the six other c. albula populations .
these patterns were confirmed by the ibd analyses, which demonstrated that increasing genetic distances corresponded to increasing geographical distances only for the six lakes with single c. albula populations.
the interruption of the ibd became obvious when all eight c. albula populations were included, suggesting that the c. albula in sympatry with spring-spawners are genetically distinct from other c. albula populations.
even within the six allopatric c. albula populations, the ibd model was slightly biased by a higher-than-expected genetic distance between the neighboring lakes ploener and behler.
this is attributable to a higher genetic similarity of the population from lake behler to the sympatric populations and to c. albula from lake arend.
the c. albula population in lake arend is not native, but has been introduced from lake enzig  about  <dig> years ago  <cit> .
historical introduction of c. albula in lake behler cannot be excluded, whereas recent stocking can be ruled out  <cit> .
altogether, the results from our analyses suggest a complex evolutionary scenario for the sympatric populations, including putative parallel sympatric speciation, introgression on secondary contact, divergent selection and within-lake hybridization.
taken together, our results suggest the following evolutionary and phylogeographical hypothesis: all lakes in northern germany including lakes stechlin and breiter luzin were invaded from a single refuge after the last glaciation about  <dig>  years ago.
this founder lineage of c. albula still exists in the majority of the lakes.
the genetic differentiation between these populations can be appropriately described by an ibd model  which is only slightly modified by more recent stocking  <cit> .
in two of the deepest lakes , parallel sympatric speciation along steep vertical abiotic gradients may have occurred.
the putative sympatric speciation is suggested by the low within-lake genetic segregation of sympatric pairs by neutral markers  <cit> .
subsequent divergent selection may have induced a stronger genetic segregation of sympatric pairs, as suggested by the aflp markers used here.
evidence for strong recent selection comes from the fact that the removal of aflp loci putatively under selection substantially reduced the genetic differentiation of the sympatric pairs.
furthermore, physiological studies suggest that ecological divergence is driven by temperature-based adaptations of the sympatric pair at least in lake stechlin  <cit> .
recent stocking from identical autumn-spawning sources  <cit>  may have slightly reduced the genetic distance between the c. albula populations in lakes stechlin and breiter luzin, as indicated by a low frequency of hybridization between these populations and c. albula from lake tollense.
secondary contact with another coregonus lineage and subsequent introgression may have further shaped the genetic signature of the sympatric populations.
introgression is more likely for the autumn-spawning c. albula because the majority of all other european populations of the c. albula complex are autumn-spawners  <cit> .
ongoing within-lake hybridization between sympatric populations, as demonstrated here by the structure analyses, may have resulted in the spreading of introgressed genetic material into both populations.
introgression after secondary contact may explain why both c. albula and the spring-spawning c. fontanae and c. lucinensis of lakes stechlin and breiter luzin were genetically distinct to the allopatric c. albula in the other german lakes.
the source of the secondary contact could be from populations living farther east.
this is suggested by the similarity between lakes stechlin and breiter luzin and the population from lake arend, which has a historical origin in poland.
brzuzan et al.
<cit>  demonstrated that two distinct lineages exist within polish c. albula populations, with one group occurring in the east, and one in central and west poland.
these authors speculated that these may have evolved by colonization from different refugia or by introgression from c. sardinella  <cit> .
interestingly, introgression from c. sardinella has also been suspected for the lake breiter luzin populations  <cit> .
an allozyme study  <cit>  found low genetic distances between c. sardinella and c. albula populations in russia.
accordingly, we cannot exclude that populations exist in east europe which are part of a separate lineage involved into the secondary contact with the german sympatric populations, thus explaining the genetic similarity between lakes arend, stechlin and breiter luzin.
we would like to emphasize that this complex scenario is hypothetical at the moment.
we cannot exclude that the sympatric populations had an allopatric divergence or originate from two different refugia  <cit> , followed by reinforcement on secondary contact in lakes stechlin and breiter luzin.
in this case, adaptive differentiation might have followed nonecological speciation  <cit> .
based on all available evidence from this and other studies, further clarification of speciation mode will require samples of the c. albula complex from several populations in poland and russia to be included in the analysis, as well as additional sympatric populations existing in russia and scandinavia  <cit> .
comparable evidence for multiple invasions as the main mechanism for adaptive divergence within single lakes was found for c. clupeaformis  <cit>  and sticklebacks  <cit>  in north america.
therefore, we cannot exclude that russian or scandinavian sympatric populations of the c. albula complex  <cit>  underwent an allopatric stage as well.
CONCLUSIONS
the effects of hybridization and stocking on the commercially important coregonus can be quantified and accounted for, thus the group remains an excellent model for the study of speciation in sympatry.
despite having used more than  <dig> marker loci in a study of  <dig> populations, our data failed to provide conclusive evidence for whether spring-spawning coregonus stem from parallel sympatric speciation.
parallel trends in niche differentiation and phenotypic differentiation are evident in both lakes with sympatric populations, but neutral genetic differentiation suggests allopatric speciation and introgression on secondary contact.
it is clear that population genetic criteria alone cannot resolve the mechanisms of speciation, and that our analysis benefited from a large spatial scale of sampling.
examination of relative differences within and among lakes has raised the possibility that multiple lineages may be present in northern germany.
as a result, phylogenetic analysis and sampling of additional lakes from potential source populations from a broad geographic range is required for an unequivocal identification of the evolutionary mechanisms and phylogeography which have led to the ecological and genetic diversity found in the c. albula complex.

 fish in temperate lakes that have been formed since the last glaciation provide good model systems for studying adaptive radiation and ecological speciation. according to the ecological theory of adaptive radiation, environmental gradients and intense competition for scarce resources lead to divergent natural selection that, in turn, produces phenotypic differentiation. reproductive isolation is thought to evolve as a by-product of divergent natural selection because traits responsible for reproductive isolation are usually adaptive. sympatric populations of north temperate freshwater fish with significant genetic differentiation and strong ecological divergence are well known in sticklebacks, rainbow smelt ), arctic char ) or sockeye salmon ). but no group of fishes is as well known for adaptive radiations and complex speciation patterns in postglacial lakes as coregonid fishes. a recent review identified six potential modes of speciation in coregonus: sympatric speciation from a single founder lineage, repeated invasions from a single lineage, hybrid swarm radiation, and three variants of allopatric speciation; complete or following secondary contact with either subsequent genomic reinforcement or with subsequent ecological reinforcement, depending on the levels of reproductive isolation between the taxa. in order to distinguish from among these potential modes of speciation, population genetic information is required in addition to phylogenetic, biogeographical, morphological and ecological data. specifically, lower genetic differentiation between sympatric than between allopatric populations provides evidence for hybrid swarm and sympatric speciation scenarios. conversely, more pronounced reproductive isolation between sympatric populations than between allopatric populations provides evidence for allopatric speciation with reinforcement. ecological diversification leading to speciation has been repeatedly suggested in coregonus; however, recent reviews have challenged the assumption that all cases of ecological segregation between sympatric populations provide valid evidence for ecological speciation. nevertheless, particularly strong support for sympatric ecological speciation can be obtained from cases of parallel ecological divergence and genetic differentiation in similar systems. further evidence for ecological speciation can be gained from genome scans that detect the same loci under selection in different systems with similar environments. in a geographical context, the genetic distances among populations of the founder lineage of sympatric species should fit to an isolation-by-distance model, whereas adaptive divergence between the sympatric populations may disrupt the ibd patterns. subsequently, sympatric populations would be genetically different but with a geographical distance of zero, thereby reducing our ability to detect significant ibd. within the coregonidae, the postglacial evolution of whitefish and c. lavaretus complexes) and the north america ciscoes are comparatively well studied. several speciation modes have been suggested for these groups, but evidence for parallel sympatric speciation from a single founder lineage is weak in any of the systems. an alternative system to study putative parallel ecological speciation can be found within populations of the baltic ciscoes complex), a group that is widespread in northern europe. sympatric populations of the c. albula complex occur in scandinavia and russia and have been reported from two deep, stratified clear-water lakes situated geographically close to one another in northern germany. in lake stechlin, the autumn-spawning c. albula coexists with the smaller, spring-spawning c. fontanae. both species are pelagic zooplanktivores, but differ substantially with respect to temperature-dependent metabolic adaptations. analysis of six microsatellite loci revealed very low levels of genetic differentiation between the sympatric populations in lake stechlin. in the nearby lake breiter luzin, two populations also coexist, between which the microsatellite differentiation was also very low. while these results suggest a sympatric origin of each spring spawner, shared mtdna haplotypes revealed lower genetic relatedness between sympatric taxa. an important advance to our understanding of the origin of the sympatric pairs could come from a genetic comparison with other allopatric populations from the same geographical area. furthermore, the few neutral microsatellite markers employed did not allow for the study of potentially parallel adaptive divergence in both lakes. accordingly, the specific speciation mode of the sympatric populations has yet to be unambiguously determined. here, we studied genetic differentiation within the c. albula complex from eight lakes using amplified fragment length polymorphism. six lakes had single, autumn-spawning c. albula populations, whereas two lakes contained c. albula together with a sympatric species. we first examined the overall genetic structure of the coregonus populations, with particular emphasis on comparing differentiation between allopatric and sympatric populations. under the scenario of sympatric speciation, we expected to find greater genetic similarity between the sympatric populations within lakes than among lakes. species that have evolved recently may not have developed full genetic incompatibilities and thus may hybridize with little intrinsic fitness loss, therefore we also tested whether ongoing hybridization between sympatric populations may blur historical differentiation. second, we fit genetic distances to a stepwise ibd model and hypothesized a disruption of ibd if sympatric populations are included. third, we calculated the probability of hybridization between sympatric populations and fish from putative stocking source populations. enhancement stocking in the commercially important coregonus lake populations is a common management practice, and our earlier work has quantified how this can modify the genetic identity of the target populations and disrupt ibd. we hypothesized that hybridization with c. albula stocked from allopatric autumn-spawning populations would be more likely for the autumn-spawning c. albula from both lakes than for the sympatric spring-spawning c. fontanae or c. lucinensis. finally, we tested for the presence of outlier loci that were shared in both sympatric pairs to find genetic evidence for parallel adaptive divergence.  all coregonus populations and all aflp loci using a multiplex aflp method we scored loci from individuals, of which were polymorphic over the populations. differentiation between the repeated samplings of populations in lake stechlin were not significantly different from zero, thus samples were pooled for each population in subsequent analyses. the percentage of polymorphic loci varied from - 92% within the populations. estimated with hs, the expected heterozygosity ranged from to, with the highest values being found in the two c. albula populations that occur with the sympatric species. lake names, geographical position and genetic diversity of the coregonus albula complex populations sampled. allopatric populations consist exclusively of c. albula. sympatric populations are named by lake origin and the lake-specific species names. n = number of individuals analysed, % polymorphic loci refers to the total of loci analysed, hs = gene diversity with credibility intervals calculated by a bayesian approach as implemented in hickory, hs = the mean within-population expected heterozygosity. there was significant genetic differentiation among the populations, with % of variation found among populations. global values of θ and θb were nearly identical and also showed significant population structure. all pairwise θ-values were significantly different from zero and ranged from to. all nodes of the nj tree of nei's genetic distances were supported by bootstrap values of at least 95%. the tree grouped four lakes with single populations as distinct from the two lakes with sympatric population pairs. lakes arend and behler appeared more closely related to stechlin and breiter luzin. interestingly, the allopatric spring-spawners, c. fontanae and c. lucinensis, grouped more closely to one another than either did to its sympatric c. albula population. pairwise genetic and geographical distances were not correlated for the entire dataset, providing evidence that an ibd model did not appropriately describe the pattern of genetic differentiation among all populations. an ibd model was also not appropriate to describe the genetic differentiation between all eight c. albula populations including those from lakes stechlin and breiter luzin. in contrast, when only the six lakes with only a c. albula population were included, genetic distances increased significantly with geographical distance as predicted by an ibd model. however, the pairwise difference between the closely located lakes ploener and behler appeared to be slightly higher than predicted by strict linear pattern. sympatric pairs lake stechlin lake stechlin genetic differentiation for the two sympatric population pairs of the coregonus albula complex. values were calculated with hickory based on all aflp loci and after removal of n = loci putatively under selection at p =. the analyses of potential gene flow within the two lakes by structure found some evidence for the existence of hybrids between sympatric populations. the mean proportion of any individual's genome that originated from its own population was generally ≥ 87%. three individuals showed elevated probabilities of misclassification or hybridization in the f1-generation. for individuals, there was a high probability of f2- or older hybridizations with the sympatric population. in contrast, the probability of f1-hybrids formed with stocked fish from lakes arend and tollense was zero for all populations, and only six individuals showed a high probability of f2- and older hybrids with stocked fish. outlier loci the explorative genome scans revealed very few loci to be positively exceeding neutral expectations in the two lakes with sympatric population pairs. more loci under putative selection were found in lake stechlin than in lake breiter luzin. no single locus was non-neutral in both lakes. at the p = quantile, % of loci were positively non-neutral in lake stechlin, and % in breiter luzin. at this quantile, three loci were shared from both lakes. after removal of all loci putatively under selection above the p = quantile in either lake stechlin or breiter luzin, genetic differentiation based on the remaining loci declined considerably, in particular between the sympatric pairs. θb-values were almost identical between c. albula and c. fontanae in lake stechlin and c. albula and c. lucinensis in lake breiter luzin, and did not differ from the differentiation between c. fontanae and c. lucinensis. only differentiation between both c. albula populations was still lower, with its credibility interval not overlapping with those of any other pairwise comparisons. genetic distances between the eight c. albula populations remained almost unaffected by the removal of outlier loci. the nj-tree calculated from the putatively neutral aflp loci had an identical topology to the nj-tree based on all aflp loci, with only minimal changes in branch lengths. discussion conflicting evidence for sympatric speciation our results suggest that the post-glacial evolution of sympatric coregonus pairs studied here is more complex than previously thought. neither spring spawner occurred elsewhere in allopatry, supporting the hypothesis that they evolved by sympatric speciation from the autumn-spawning c. albula within each lake, as suggested previously. nonetheless, the pattern of genetic differentiation that we observed contradicts this hypothesis. in cases of sympatric speciation, sympatric taxa should be more closely related, but here the autumn-spawning c. albula were more similar to each other than either was to its sympatric spring spawner. this was evident from the tests of pairwise population differentiation as well as from the nj-tree based on genetic distance. these results were similar to an allozyme study of finnish lakes, where allopatric autumn spawners formed a separate group to winter- or spring spawners. the complexity of the evolutionary history is evidenced by the somewhat conflicting results of previous genetic analyses. six microsatellite loci revealed greater genetic similarity between sympatric spring- and autumn-spawners within lakes, thus suggesting ongoing gene flow and a sympatric origin. in contrast, mtdna from the same study qualitatively support the aflp results here, that differentiation between sympatric taxa is greater than between allopatric taxa. in particular, the two most abundant of four recorded haplotypes were shared by c. albula in both lakes. one of these was shared with the stechlin sympatric c. fontanae and the other was shared with the lake breiter luzin sympatric c. lucinensis. c. fontanae and c. lucinensis shared no haplotypes. the reason for the discrepancy between the microsatellite and aflp data in one of the population pairs is not clear. the large number of aflp loci employed here should provide a better estimate of divergence than microsatellite loci, although we found evidence that not all loci were strictly neutral. the two marker systems produced similar estimates of genetic structure for populations of brown trout in norwegian lakes, although admixture was greater based on aflp. aflp results were more congruent than mtdna with morphological characters in mormyrid electric fishes, likely due to mitochondrial introgression among lineages. accordingly, aflp seem to be an appropriate tool to examine the genetic differentiation of incipient ecological speciation. but as noted above, our results suggest that not all aflp markers were strictly neutral. removing the loci putatively under selection reduced the within-lake genetic distances considerably, and differentiation based on the remaining neutral aflp loci was more similar to the pattern obtained from the neutral microsatellite data. interestingly, the genetic distance to other, allopatric c. albula populations remained largely unaffected. previous studies similarly found that differentiation between ecologically or morphologically distinct populations can be maintained for a small portion of the genome, whereas gene exchange continues to prevent divergence at the majority of loci. only three outlier loci occurred in both sympatric pairs at p >. it is possible that such a small number of loci positively exceeding neutral expectations might have occurred just by chance, thus providing little evidence in support of the hypothesis that the sympatric pairs result from parallel sympatric speciation. alternatively, it may be that only a few genes are involved. the few shared loci deserve further attention since recent analyses in the c. clupeaformis complex in north america indicated that these θ outliers can be significantly clustered around quantitative trait loci involved in ecologically based reproductive isolation. proportions of outlier loci substantially exceeding 1% were found in other studies applying a similar pairwise approach. the presumed modes of ecological segregation have been well studied in lake stechlin, where sympatric taxa perform diel vertical migrations with different amplitudes and they differ in their final temperature preferendum and their metabolic costs of swimming. the commonly observed benthic-limnetic habitat and diet segregations typical for whitefish do not occur in stechlin. such a pattern of physiological segregation but low trophic differentiation presumably exists for the species pair in lake breiter luzin, since both lakes are deep, with steep temperature gradients and overall low productivity. recent data suggest c. albula and c. lucinensis in breiter luzin have similar diets, as in lake stechlin. however, adaptive divergence may be weaker in lake breiter luzin than in lake stechlin. for example, c. lucinensis and c. albula do not show the same strong vertical microhabitat segregation as found between c. fontanae and c. albula in lake stechlin. a weaker adaptive divergence is supported by the lower genetic distance and a substantially lower number of loci putatively under selection in lake breiter luzin than in lake stechlin. accordingly, these species pairs would resemble'isolation by adaptation', a process in which levels of genetic differentiation among population pairs will be positively correlated with degree of adaptive phenotypic divergence. stocking and hybridization an important part of our analysis was to determine whether the genetic similarity of stechlin and breiter luzin c. albula was a result of modification by stocking and hybridization. enhancement stocking of vendace populations by freshly hatched larvae obtained primarily from two source populations has been practised in germany for at least years, with the aim of stabilizing natural recruitment and fisheries yield of sympatric and allopatric c. albula populations. because both putative stocking sources contain only autumn-spawning c. albula populations, hybridization with c. albula in stechlin and breiter luzin was hypothesized to be more likely than hybridization with the spring-spawning c. fontanae and c. lucinensis. our results support this hypothesis. furthermore, our recent analysis of the genetic effects of stocking on allopatric c. albula populations revealed a reduction of genetic distance by about 10% between lakes stocked intensively with a genetically similar source. accordingly, a weak secondary homogenization between lakes stechlin and breiter luzin by stocking from lakes arend or tollense cannot be ruled out, but the evidence is not strong enough to conclude that the similarity of the autumn spawners is an exclusive effect of current fisheries management practices. hybridization within the lakes could also contribute to the observed patterns, whereby introgression of c. albula genetic polymorphisms may obscure any historical similarities of the two spring spawners. because species that have evolved since the most recent glaciation about years ago may not have developed full genetic incompatibilities, they may hybridize with little fitness loss. the probability that single individuals have parents or grandparents from the other population was low, but greater than zero in all four populations. therefore, gene flow between the sympatric populations may not be completely interrupted, despite the differing spawning times. weak ongoing hybridization between the sympatric populations can also be inferred from the higher heterozygosity of sympatric compared with allopatric c. albula populations. in this analysis, we used a high prior probability of pure ancestry similarly applied to the analysis of sympatric whitefish populations from the canadian cliff lake. this sympatric whitefish population pair was identified as being reproductively isolated by aflp-markers, but nevertheless encompassed seven out of individuals with strong evidence for mixed ancestry. accordingly, occasional hybridization within the two sympatric populations of the c. albula complex studied here cannot be excluded. large-scale genetic structure of coregonus an important finding was that the sympatric populations of lakes stechlin and breiter luzin were genetically distinct from the six other c. albula populations. these patterns were confirmed by the ibd analyses, which demonstrated that increasing genetic distances corresponded to increasing geographical distances only for the six lakes with single c. albula populations. the interruption of the ibd became obvious when all eight c. albula populations were included, suggesting that the c. albula in sympatry with spring-spawners are genetically distinct from other c. albula populations. even within the six allopatric c. albula populations, the ibd model was slightly biased by a higher-than-expected genetic distance between the neighboring lakes ploener and behler. this is attributable to a higher genetic similarity of the population from lake behler to the sympatric populations and to c. albula from lake arend. the c. albula population in lake arend is not native, but has been introduced from lake enzig about years ago. historical introduction of c. albula in lake behler cannot be excluded, whereas recent stocking can be ruled out. altogether, the results from our analyses suggest a complex evolutionary scenario for the sympatric populations, including putative parallel sympatric speciation, introgression on secondary contact, divergent selection and within-lake hybridization. taken together, our results suggest the following evolutionary and phylogeographical hypothesis: all lakes in northern germany including lakes stechlin and breiter luzin were invaded from a single refuge after the last glaciation about years ago. this founder lineage of c. albula still exists in the majority of the lakes. the genetic differentiation between these populations can be appropriately described by an ibd model which is only slightly modified by more recent stocking. in two of the deepest lakes, parallel sympatric speciation along steep vertical abiotic gradients may have occurred. the putative sympatric speciation is suggested by the low within-lake genetic segregation of sympatric pairs by neutral markers. subsequent divergent selection may have induced a stronger genetic segregation of sympatric pairs, as suggested by the aflp markers used here. evidence for strong recent selection comes from the fact that the removal of aflp loci putatively under selection substantially reduced the genetic differentiation of the sympatric pairs. furthermore, physiological studies suggest that ecological divergence is driven by temperature-based adaptations of the sympatric pair at least in lake stechlin. recent stocking from identical autumn-spawning sources may have slightly reduced the genetic distance between the c. albula populations in lakes stechlin and breiter luzin, as indicated by a low frequency of hybridization between these populations and c. albula from lake tollense. secondary contact with another coregonus lineage and subsequent introgression may have further shaped the genetic signature of the sympatric populations. introgression is more likely for the autumn-spawning c. albula because the majority of all other european populations of the c. albula complex are autumn-spawners. ongoing within-lake hybridization between sympatric populations, as demonstrated here by the structure analyses, may have resulted in the spreading of introgressed genetic material into both populations. introgression after secondary contact may explain why both c. albula and the spring-spawning c. fontanae and c. lucinensis of lakes stechlin and breiter luzin were genetically distinct to the allopatric c. albula in the other german lakes. the source of the secondary contact could be from populations living farther east. this is suggested by the similarity between lakes stechlin and breiter luzin and the population from lake arend, which has a historical origin in poland. brzuzan et al. demonstrated that two distinct lineages exist within polish c. albula populations, with one group occurring in the east, and one in central and west poland. these authors speculated that these may have evolved by colonization from different refugia or by introgression from c. sardinella. interestingly, introgression from c. sardinella has also been suspected for the lake breiter luzin populations. an allozyme study found low genetic distances between c. sardinella and c. albula populations in russia. accordingly, we cannot exclude that populations exist in east europe which are part of a separate lineage involved into the secondary contact with the german sympatric populations, thus explaining the genetic similarity between lakes arend, stechlin and breiter luzin. we would like to emphasize that this complex scenario is hypothetical at the moment. we cannot exclude that the sympatric populations had an allopatric divergence or originate from two different refugia, followed by reinforcement on secondary contact in lakes stechlin and breiter luzin. in this case, adaptive differentiation might have followed nonecological speciation. based on all available evidence from this and other studies, further clarification of speciation mode will require samples of the c. albula complex from several populations in poland and russia to be included in the analysis, as well as additional sympatric populations existing in russia and scandinavia. comparable evidence for multiple invasions as the main mechanism for adaptive divergence within single lakes was found for c. clupeaformis and sticklebacks in north america. therefore, we cannot exclude that russian or scandinavian sympatric populations of the c. albula complex underwent an allopatric stage as well.  the effects of hybridization and stocking on the commercially important coregonus can be quantified and accounted for, thus the group remains an excellent model for the study of speciation in sympatry. despite having used more than marker loci in a study of populations, our data failed to provide conclusive evidence for whether spring-spawning coregonus stem from parallel sympatric speciation. parallel trends in niche differentiation and phenotypic differentiation are evident in both lakes with sympatric populations, but neutral genetic differentiation suggests allopatric speciation and introgression on secondary contact. it is clear that population genetic criteria alone cannot resolve the mechanisms of speciation, and that our analysis benefited from a large spatial scale of sampling. examination of relative differences within and among lakes has raised the possibility that multiple lineages may be present in northern germany. as a result, phylogenetic analysis and sampling of additional lakes from potential source populations from a broad geographic range is required for an unequivocal identification of the evolutionary mechanisms and phylogeography which have led to the ecological and genetic diversity found in the c. albula complex. 
sympatric pairs in the two lakes were genetically distinct from the six other c. albula populations, suggesting introgression from another lineage may have influenced these two lakes. here, we analysed the genetic structure of baltic ciscoes, examining individuals from lakes in northern germany using polymorphic aflp loci. relative differences within and among lakes raises the possibility that multiple lineages may be present in northern germany, thus understanding the postglacial evolution and speciation in the c. albula complex requires a large-scale phylogenetic analysis of several potential founder lineages. however, removal of all non-neutral loci decreased the genetic distance between sympatric pairs, suggesting recent adaptive divergence at a few loci. this was supported by an analysis of isolation-by-distance, where the drift-gene flow equilibrium observed among allopatric populations was disrupted when the sympatric pairs were included. while the population genetic data alone can not unambiguously uncover the mode of speciation, our data indicate that multiple lineages may be responsible for the complex patterns typically observed in coregonus. genome scans found only three loci that appeared to be under selection in both sympatric population pairs, suggesting a low probability of similar mechanisms of ecological segregation. of particular interest is whether the repeated occurrence of sympatric species pairs results from in-situ divergence from a single lineage or from multiple invasions of one or more different lineages. six lakes had only one population of c. albula while the remaining two lakes had c. albula as well as a sympatric species. teleost fishes of the coregonidae are good model systems for studying postglacial evolution, adaptive radiation and ecological speciation. aflp demonstrated a significant population structure. lower differentiation between allopatric than sympatric populations contradicts the hypothesis of a sympatric origin of taxa, and there was little evidence for stocking or ongoing hybridization.
 teleost fishes of the coregonidae are good model systems for studying postglacial evolution, adaptive radiation and ecological speciation. of particular interest is whether the repeated occurrence of sympatric species pairs results from in-situ divergence from a single lineage or from multiple invasions of one or more different lineages. here, we analysed the genetic structure of baltic ciscoes, examining individuals from lakes in northern germany using polymorphic aflp loci. six lakes had only one population of c. albula while the remaining two lakes had c. albula as well as a sympatric species.  aflp demonstrated a significant population structure. lower differentiation between allopatric than sympatric populations contradicts the hypothesis of a sympatric origin of taxa, and there was little evidence for stocking or ongoing hybridization. genome scans found only three loci that appeared to be under selection in both sympatric population pairs, suggesting a low probability of similar mechanisms of ecological segregation. however, removal of all non-neutral loci decreased the genetic distance between sympatric pairs, suggesting recent adaptive divergence at a few loci. sympatric pairs in the two lakes were genetically distinct from the six other c. albula populations, suggesting introgression from another lineage may have influenced these two lakes. this was supported by an analysis of isolation-by-distance, where the drift-gene flow equilibrium observed among allopatric populations was disrupted when the sympatric pairs were included.  while the population genetic data alone can not unambiguously uncover the mode of speciation, our data indicate that multiple lineages may be responsible for the complex patterns typically observed in coregonus. relative differences within and among lakes raises the possibility that multiple lineages may be present in northern germany, thus understanding the postglacial evolution and speciation in the c. albula complex requires a large-scale phylogenetic analysis of several potential founder lineages. 