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doi: 10.1111/j.1365-294X.2008.03714.x
H . S . M K I N E N , J . M . C A N O and J . M E R I L
Ecological Genetics Research Unit, Department of Biological and Environmental Sciences, PO Box 65, FI-00014 University of Helsinki,
Helsinki, Finland
Abstract
Natural selection is expected to leave an imprint on the neutral polymorphisms at the adjacent
genomic regions of a selected gene. While directional selection tends to reduce withinpopulation genetic diversity and increase among-population differentiation, the reverse
is expected under balancing selection. To identify targets of natural selection in the
three-spined stickleback (Gasterosteus aculeatus) genome, 103 microsatellite and two
indel markers including expressed sequence tags (EST) and quantitative trait loci (QTL)associated loci, were genotyped in four freshwater and three marine populations. The
results indicated that a high proportion of loci (14.7%) might be affected by balancing selection
and a lower proportion (2.8%) by directional selection. The strongest signatures of
directional selection were detected in a microsatellite locus and two indel markers located
in the intronic regions of the Eda-gene coding for the number of lateral plates. Yet, other
microsatellite loci previously found to be informative in QTL-mapping studies revealed no
signatures of selection. Two novel microsatellite loci (Stn12 and Stn90) located in chromosomes I and VIII, respectively, showed signals of directional selection and might be linked
to genomic regions containing gene(s) important for adaptive divergence. Although the
coverage of the total genomic content was relatively low, the predominance of balancing
selection signals is in agreement with the contention that balancing, rather than directional
selection is the predominant mode of selection in the wild.
Keywords: balancing selection, directional selection, Gasterosteus aculeatus, genome scan, hitchhiking,
microsatellite
Received 25 October 2007; revision accepted 22 January 2008
Introduction
The amount of sequence information available for various
organisms has recently increased tremendously (Benson
et al. 2007), but the functional properties of the sequence
data remain mostly unresolved. For example, the identity
of genes underlying evolutionary change is still largely
unknown especially in wild populations (e.g. Mackay 2001;
Orr 2005a, but see Orr 2005b). One promising approach to
locate genes involved in adaptation is to use the hitchhikingmapping approach to detect genomic regions showing
footprints of natural selection (Schltterer 2003; Storz 2005;
Vasemgi & Primmer 2005). Population genetic theory
Correspondence: H. S. Mkinen, Fax: +358-9-191 57694; E-mail:
hannu.makinen@helsinki.fi
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3566 H . S . M K I N E N , J . M . C A N O and J . M E R I L
Teshima et al. 2006). Especially in single-locus studies,
population expansions or bottlenecks can have a similar
effect on neutral polymorphisms as expected under selection (Simonsen et al. 1995). However, selection will affect only
locus-specific patterns in neutral polymorphisms as compared to the genome-wide effects of population history and
demographic events. Consequently, genotyping a large
number of neutral polymorphisms scattered throughout
the organisms genome is an effective way to tell apart the
effect of selection from the confounding effects of population
history (Schltterer 2003).
Empirical and theoretical studies have shown that the
probability to detect signatures of selection depends largely
on the chromosomal distance between the selected site and
a neighbouring locus, as well as on the strength of selection
(Storz 2005; Cano et al. 2006; De Kovel 2006). The signal of
selection decays as recombination breaks down the linkage
disequilibrium between the selected site and a linked
locus. In wild populations, the footprint of directional
selection can be lost when many generations have elapsed
since the selection event (Storz 2005; De Kovel 2006). Therefore, tight linkage between the marker locus and the actual
target of selection would improve the probability to detect
targets of natural selection. Utilizing molecular markers
such as microsatellites found within expressed sequence
tags (EST) would be a logical starting point for the screening of gene-associated polymorphisms (Vasemgi et al.
2005).
Northern Hemisphere fish populations are suitable
models for screening of genomic regions underlying adaptation. During the northward recolonization after the last
glaciation (c. 10 000 years ago) different fish species adapted
to numerous newly formed freshwater habitats, leading to
rapid phenotypic diversification (Taylor & McPhail 2000;
stbye et al. 2006; Rogers & Bernatchez 2007). For example,
in the Fennoscandian Atlantic salmon populations several
genomic regions showed indications of divergent selection
as a result of adaptation to the salt, brackish and freshwater
habitats (Vasemgi et al. 2005). Furthermore, comparisons
between quantitative genetic and neutral genetic differentiation have shown that the phenotypic differences
might have been shaped by directional selection even in
very short timescales (Leinonen et al. 2008).
Fennoscandian three-spined stickleback (Gasterosteus
aculeatus) populations provide an excellent system for
genome scan studies. Marine three-spined sticklebacks have
post-glacially colonized numerous freshwater habitats in
Fennoscandia showing extensive adaptive divergence in
morphological traits (Cano et al. 2006; Leinonen et al. 2006;
Mkinen et al. 2006) as has been documented for the species
throughout its distribution range (Bell & Foster 1994;
McKinnon & Rundle 2002). In addition, there is a large
amount of genomic information available on the genetic
basis of the morphological differentiation in three-spined
N AT U R A L S E L E C T I O N I N S T I C K L E B A C K S 3567
Fig. 1 The geographical locations of the six
Scandinavian study populations are shown
in the larger map and the distantly located
R. Neretva population in the inset map. The
plate morphs are indicated next to the
population labels.
Table 1 Basic information of the sample sites and genetic diversity (HE), allelic richness (AR) and inbreeding coefficient (FIS) estimates
Population
Coordinates
Drainage
Habitat
Plate morph
HE
AR
FIS
Merirastila
Orrevatnet
Barents
L. Vttern
L. Pulmanki
L. Kevo
R. Neretva
6010N, 2500E
6019N, 0522E
7458N, 3708E
5854N, 1424E
6958N, 2758E
6945N, 2700E
4306N, 1743E
Baltic Sea
North Sea
Barents Sea
S Sweden/Baltic Sea
N Finland/Barents Sea
N Finland/Barents Sea
W Bosnia/Adriatic Sea
Marine (coastal)
Marine (coastal)
Marine (pelagic)
Freshwater (lake)
Freshwater (lake)
Freshwater (lake)
Freshwater (river)
Full
Full
Full
Partial
Low
Full
Low
0.76
0.66
0.73
0.74
0.67
0.55
0.58
8.8
5.1
7.7
8.0
6.1
5.2
5.7
0.028
0.007
0.011
0.033
0.0
0.026
0.026
3568 H . S . M K I N E N , J . M . C A N O and J . M E R I L
Fig. 2 A physical map of 21 three-spined stickleback chromosomes showing the genomic positions of the microsatellite loci used in this
study. The map was depicted from the genome sequence available at the http://www.ensembl.org/Gasterosteus_aculeatus/index.html.
The positions of the QTLs from published studies are shown along the chromosomes (Peichel et al. 2001; Colosimo et al. 2004, 2005; Shapiro
et al. 2004; Kimmel et al. 2005). Note that the position of the markers and QTLs may have changed in comparison to the original linkage map
(Peichel et al. 2001).
N AT U R A L S E L E C T I O N I N S T I C K L E B A C K S 3569
ESTs, 16 closely linked to ESTs (within a 50 kb window)
and seven loci were located in noncoding DNA. To look for
putative homologies of the candidate genes, a protein
protein blast search was conducted on NCBI using the
protein sequence from Ensembl transcript predictions.
The biological processes of the putative homologies were
classified according to the gene ontology (GO) categories
(Harris et al. 2006).
Data analysis
Basic population genetic parameters, such as expected heterozygosity, allelic richness and deviations from the Hardy
Weinberg equilibrium were calculated as implemented in
the fstat 2.9.3.2 (Goudet 2001). Population differentiation
was calculated using the estimator (Weir & Cockerham
1984) and the 95% confidence intervals were determined
by 1000 permutations. To test whether stepwise mutations
had contributed to the population differentiation in addition
to random genetic drift, an allele permutation test was
applied as implemented in the program spagedi (Hardy
& Vekemans 2002; Hardy et al. 2003). Analysis of molecular
variance was used to partition the genetic variation
between-groups, among-populations and within-population
components and was calculated with arlequin 3.1
(Schneider et al. 2000).
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3570 H . S . M K I N E N , J . M . C A N O and J . M E R I L
increase of false positives rather than detecting new candidate loci under selection (Beaumont & Balding 2004).
The Bayesian FST test was carried out across all study
populations and between populations within habitat types
(marine or freshwater; Table 1). Also, comparisons between
plate morphs (fully vs. low/partially plated; Table 1) were
conducted. Two thousand draws from the posterior probability distribution were used to summarize the parameter
estimates for the locus effects () and were calculated in the
R package (http://www.r.project.org/) using the functions
provided with the distribution package of bayesfst (http://
www.reading.ac.uk/Statistics/genetics/software.html).
Two independent runs starting from different parameter
values were performed to check whether the MCMCsimulation converged to similar parameter estimates.
To further investigate the outlier loci detected by the
Bayesian FST test an analysis of molecular variance (amova)
was carried out in arlequin 3.1 (Schneider et al. 2000). Here,
a simple working hypothesis was assumed: if the loci are
affected by directional selection, their among-population
variance component should exceed the variance observed
in neutral loci. In a similar way, if a locus is affected by
balancing selection then the variance among populations
should be lower than in the neutral loci. The analysis was
conducted separately for the putatively neutral, directional
and balancing selection loci classified a posteriori according
to the Bayesian FST test. The populations were arranged
in freshwater and marine groups to analyse the effect of
habitat type on the partition of the total variance.
The Ln RH test was used in the pairwise comparisons to
detect signatures of directional selection (Kauer et al. 2003).
In this analysis the primary focus was to get additional
evidence for directional selection for the loci identified in
the Bayesian FST test. The Ln RH test is based on the
assumption that directional selection decreases the genetic
diversity ( = 4Ne) around the selected site in comparison
to the genome-wide effects of the population structure and
history (Kauer et al. 2003). Standardized (mean = 0, SD = 1)
Ln RH estimates are expected to be normally distributed
under neutrality and 95% of the values are expected to fall
between 1.96 and 1.96 (Kauer et al. 2003). Thus, loci with
Ln RH estimates outside these boundaries were considered
significant at the 5% level. The power of this test is assumed
to be higher if compared populations differ in genetic
diversity levels, i.e. in cases where only the other population
is experiencing directional selection (Storz 2005). All possible
pairwise comparisons were carried out within- and betweenhabitat types (i.e. marine and freshwater). In addition,
comparisons between low and full plated populations
were conducted. In order to estimate the Ln RH statistics,
the genetic diversity () was calculated as implemented in
the constrained gene diversity option in the program
microsatellite analyser (Dieringer & Schltterer 2003).
Another test statistics, Ln RV, which is based on the relative
Results
Basic population genetic structure
The level of polymorphism varied considerably among the
105 studied loci. The expected heterozygosities ranged
from 0.08 (GAest32) to 0.98 (GAest30) and the number of
alleles from two (Stn381) to 78 (GAest30). The average
expected heterozygosity was 0.79 and the number of alleles
20.3. Four loci and two populations (Merirastila and L.
Vttern) deviated from the HardyWeinberg expectations
showing a slight excess of homozygosity, but this effect
disappeared after correcting for multiple tests at an -level
of 0.05 (Table 1). A comparison between habitat types
revealed similar average allelic richness in marine (7.2) and
in freshwater populations (6.3) (two-tailed permutation
test, P = 0.44). Likewise, expected heterozygosity was similar
in marine (0.72) and in freshwater populations (0.66,
P = 0.23). Genetic differentiation (FST) among marine
populations was markedly lower (0.06) than in freshwater
populations (0.23) but again not significant (P = 0.13). The
amova analysis indicated a negligible effect of habitat type
(0.88%, 1000 permutations; P = 0.16), whereas most of
the variation was explained by the among-population
component (16.07%, P < 0.001) and the within-population
component (83.05%, P < 0.0001; Table 3). The estimate of
the FST according to Weir & Cockerham (1984) across all
populations indicated moderate genetic differentiation
(FST = 0.167, 95% CI; 0.1480.189). There was considerable
heterogeneity in the locus-specific FSTs ranging from 0.037
(GAest32) to 0.859 (Stn381).
N AT U R A L S E L E C T I O N I N S T I C K L E B A C K S 3571
Fig. 3 (ae) Summary of the Bayesian FST method results depicting the comparisons between different combinations of populations. The
locus effects () are plotted against their P-values. The solid line indicates the critical P-value for the directional selection and the dashed
line indicates the corresponding cut-off for the balancing selection loci. The P-values (x-axis) were transformed as logit (2|P 0.5|) for a
better visualization.
There was a considerable overlap in the number and identities of loci under balancing selection in the global and
freshwater comparisons. Fifteen loci were detected as balancing selection outliers in the global analysis, and 16 loci in
the analysis including only the freshwater populations.
The number of false positives was higher in the freshwater
comparison (six loci) as compared to the global comparison
(three loci, Table 4). Two of the loci were not the same as in
the global comparison (GAest36 and GAest57) and three
additional loci (Stn163, Stn208 and GAest42) were
unique to the freshwater populations (Table 4, Fig. 3a, b).
No evidence for directional selection was found within the
marine populations and only two loci (GAest30 and
GAest60) appeared to be affected by balancing selection
(Fig. 3c). Therefore, most of the signatures of selection were
3572 H . S . M K I N E N , J . M . C A N O and J . M E R I L
Table 2 The mean degree of population differentiation (FST),
expected heterozygosity (HE) and allelic richness (AR) of putatively
neutral, directional and balancing selection loci as identified in
the Bayesian FST method in the global comparison. Numbers in
parentheses refer to the number of loci in each category
FST
HE
AR
Neutral (85)
Directional (5)
Balancing (15)
0.11
0.77
6.3
0.40
0.63
2.8
0.04
0.94
11.7
Discussion
This study revealed novel regions in the three-spined
stickleback genome that may have been affected by natural
selection. The majority of the selective footprints were
caused by the patterns of allelic divergence among the
freshwater populations, which is consistent with existing
knowledge about phenotypic adaptive divergence observed
Table 3 Analysis of molecular variance (amova) showing the partition microsatellite allele frequencies to the among-group (habitat type),
among-populations within groups and within-population variance components for the putatively neutral, directional and balancing
selection loci identified from the Bayesian FST test. The number of loci is given in the parenthesis
Directional (5)
Balancing (15)
Neutral (85)
All loci (105)
Among-groups
FCT
Among-populations
FSC
Within-populations
FST
26.9
0.0
0.0
0.88
0.27*
0.0
0.0
0.009
37.5
6.7
16.6
16.1
0.51***
0.07***
0.17***
0.16***
35.6
93.4
83.8
83.1
0.64***
0.07***
0.16***
0.17***
Table 4 Summary of the outlier loci detected by the Bayesian FST method in the global and freshwater comparisons. The putative homologies of the outlier loci and the biological processes
according to the GO categories. The P-values have to be interpreted as the quantilies of the posterior distribution of the locus effect (Beaumont & Balding 2004). In other words, P is
significantly positive if its 2.5% quantile is positive, and is significantly negative if its 97.5% quantile is negative. The q-values are given in parenthesis
Global
Freshwater
P (q-value)
Biological process
Protein homology
Stn12
Stn90
Stn365
Stn380
Stn381
GAest84
GAest87
0.32
0.40
0.37
0.42
0.46
0.28
0.28
0.007 (0.032)
0.0005 (0.004)
0.014 (0.043)
0.0015 (0.009)
0.0025 (0.015)
0.02 (0.057)
0.012 (0.057)
Unknown
Regulation of transcription
Lateral plate #
Lateral plate #
Lateral plate #
Unknown
Unknown
0.996 (0.981)
0.995 (0.977)
Stn122
Stn19
0.077
0.071
0.978 (0.942)
0.993 (0.967)
0.037
0.046
0.044
0.034
0.038
0.042
0.024
0.055
0.059
0.999 (0.998)
0.997 (0.985)
0.994 (0.975)
0.999 (0.998)
0.999 (0.996)
0.998 (0.991)
0.999 (0.998)
0.984 (0.944)
0.982 (0.941)
Stn34
Stn57
Stn67
Stn59
Stn81
GAest8
GAest30
GAest52
0.060
0.065
0.069
0.051
0.046
0.050
0.038
0.055
0.999 (0.993)
0.992 (0.953)
0.987 (0.965)
0.999 (0.996)
0.999 (0.996)
0.999 (0.996)
0.999 (0.996)
0.999 (0.993)
GAest57
GAest60
GAest63
0.057
0.037
0.037
0.982 (0.941)
0.998 (0.991)
0.999 (0.998)
GAest60
GAest63
0.079
0.047
0.986 (0.957)
0.999 (0.996)
GAest74
0.038
0.999 (0.998)
GAest74
Stn163
Stn208
GAest42
0.052
0.072
0.078
0.070
0.999 (0.996)
0.987 (0.957)
0.978 (0.943)
0.988
Cell-Cell signalling
Multicellular
organismal development
Protein binding
Vesicle mediated transport
Unknown
Unknown
Unknown
Unknown
Unknown
Unknown
Brain segmentation,
hindbrain development
Unknown
Unknown
UDP-N-acetylgalactosamine
metabolism
Unknown
Unknown
Unknown
carboxylic acid metabolism
P (q-value)
Directional
Stn12
Stn90
Stn365
Stn380
Stn381
0.19
0.28
0.42
0.52
0.56
0.016 (0.056)
0.002 (0.011)
0.0003 (0.002)
0.0003 (0.002)
0.0003 (0.002)
Balancing
Stn122
Stn19
0.050
0.054
Stn34
Stn57
Stn67
Stn59
Stn81
GAest8
GAest30
GAest36
GAest52
N AT U R A L S E L E C T I O N I N S T I C K L E B A C K S 3573
Bayes FST
Bayes FST
3574 H . S . M K I N E N , J . M . C A N O and J . M E R I L
Table 5 Pairwise Ln RH comparisons indicating significant loci and the LnRH estimates in parenthesis
Merirastila
Orrevatnet
Barents
L. Pulmanki
L. Kevo
L. Vttern
R. Neretva
Merirastila
Orrevatnet
Barents
L. Pulmanki
NA
Stn12 (2.01)
Stn90 (2.54)
NA
Stn12 (2.16)
Stn90 (2.0)
NA
L. Kevo
NA
Stn12 (2.46)
Stn365 (2.15)
Stn 90 (2.33)
GAest84 (2.37)
NA
L. Vttern
Stn380 (2.40)
Stn12 (1.98)
Stn380 (2.58)
GAest84 (2.02)
Stn380 (2.27)
Stn12 (2.05)
Stn90 (2.36)
Stn365 (2.36)
Stn90 (2.73)
NA
Stn380 (2.57)
Stn380 (2.82)
Stn380 (2.73)
NA
R. Neretva
N AT U R A L S E L E C T I O N I N S T I C K L E B A C K S 3575
than directional selection signals in allozyme data sets in
the rodent genus Peromyscus. The contention that balancing
selection is more common than directional selection in
three-spined sticklebacks should be considered as preliminary given the low genomic coverage of this study, as
well as the methodological limitations discussed below. A
more dense genomic coverage and replicate populations
would be needed to confirm the generality of this pattern.
It has been also suggested that balancing selection is
responsible for maintaining polymorphism at selected loci
(Charlesworth 2006). Some recent studies, however, have
reported a limited role of balancing selection in preserving
trans-species level polymorphisms (Asthana et al. 2005).
In humans Bubb et al. (2006) found 16 high diversity SNP
regions but the distribution fell within the neutral expectations. A classical example of balancing selection maintaining
high genetic diversity is the major histocompatibility
(MHC) loci, which play a key role in vertebrate immune
defence (e.g. Garrigan & Hedrick 2003). The high number
of alleles is expected to be maintained due to overdominance (heterozygote advantage) or frequency dependent
selection (rare allele advantage). An attempt to demonstrate balancing selection operating in MHC-linked
microsatellites failed in wild sheep (Ovis dalli), probably due
to recombination breaking down the linkage between the
markers and causative polymorphisms (Worley et al. 2006).
In this study, the biological and molecular function of the
majority of the genomic regions underlying balancing
selection remains unclear since no putative homologies to
known genes were found.
Detecting balancing selection with the Bayesian FST
method is not without limitations. First of all, it has a low
power to detect balancing selection from simulated data
sets, but, on the other hand, the false discovery rate is
extremely low (0.01%, Beaumont & Balding 2004). This
may indicate that when balancing selection loci are detected,
the signal is probably very strong. Secondly, FST tends to
underestimate the degree of genetic differentiation in
highly polymorphic loci (Hedrick 2005). This was also
evident in our data; there was a negative correlation with
FST and the allelic richness across different loci (results not
shown). However, the classification of loci as indicative of
balancing selection is not solely based on the allelic richness, but also on the unusually similar allele frequencies
over populations. A third explanation for balancing selection signatures can be found from the rates of mutation. If
some microsatellite loci have a higher than average mutation rate, a higher polymorphism level could be explained
without invoking balancing selection as a cause. Although
information about the mutation rates is lacking, comparing
the FST and RST estimates on the degree of population
differentiation would be informative for the impact of
stepwise mutations. However, RST values for the candidate
loci under balancing selection did not consistently exceeded
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3576 H . S . M K I N E N , J . M . C A N O and J . M E R I L
in the Eda-gene found for the same set of populations
(Cano et al. 2006). The signal of directional selection among
the freshwater populations in the Eda-linked markers was
caused by the inclusion of an exceptional full plated L.
Kevo population in the analysis. This result contrasts with
the findings of Raeymaekers et al. 2007), suggesting that
balancing selection could explain the observed plate morph
pattern within freshwater populations in western Europe.
The results of the amova analyses and Bayesian FST test
indicate that the novel putatively selected loci are not related
to plate morph divergence but a moderate proportion of
the allele frequency variation in Stn90 was related to
differences between marine and freshwater populations.
The interpretation that Stn90 may be related to adaptation
to freshwater is only tentative at this stage; denser
mapping around this locus is needed to narrow down any
potential gene and its functional role. The other candidate
locus for directional selection, Stn12, did not show any
significant relation to plate morph but a weak effect of
habitat type. Even if it was linked to a selected gene, the
weak signal of Stn12 suggests that may be quite far from
the actual target of selection. All in all, among our set of
loci, Stn90 is the more promising candidate to find novel
genes of adaptive relevance.
N AT U R A L S E L E C T I O N I N S T I C K L E B A C K S 3577
the number of false positives is to genotype more marker
loci from the flanking regions of the candidates identified
in the initial analysis (Wiehe et al. 2007).
The FST-based and heterozygosity-ratio test results were
not fully congruent especially in the case of the microsatellite
locus and the two indel markers located in the intronic
regions of the Eda-gene. This is not surprising since the
overall heterozygosities were low at the Eda-linked loci in
most of the population comparisons and almost fixed for
different alleles. For example, the microsatellite locus Stn365
had only three alleles and the gene diversities ranged from
0 to 0.41 but the allele frequencies were highly skewed
(Appendix 4). In this situation the ratio of gene diversities
may not be informative but results in a higher than average
FST. Thus, rather than relying on only one test statistic, a
combination of allele frequency and gene diversity-ratio
based methods would exploit the information of genome
scan data sets effectively. It has been also suggested that
FST-based tests might detect selection in longer periods
after the selection event since mutation might restore the
genetic diversity to the levels before selection (Storz 2005).
Finally, if a large number of loci are selected in the compared
populations, then the standard deviation of the heterozygosity ratio becomes large and only extreme values
are significant (Storz 2005).
Conclusions
The relatively large number of genomic regions showing
footprints of balancing rather than directional selection
might be indicative of a predominant role of balancing
over directional selection in shaping the variability in the
three-spined stickleback genome. However, despite the
conservative nature of the method used to detect balancing
selection, the low genome coverage of the markers used and
the uncertainty about possible effects of differing mutation
rates among the microsatellite loci call for further studies to
verify this conclusion. The result that the number of loci
under directional selection was considerably higher among
the freshwater than among the marine populations suggests
that colonization of freshwater environments entails a
major change in selective regime. Shifting from the original
marine environment to the freshwater probably leads
to detectable imprints of selection at the genomic level.
However, it appears that balancing selection among the
freshwater populations may maintain similar allele frequencies in substantial number of loci. The outlier loci
detected in this study provide a good starting point for a
more fine-scale mapping of selective footprints. Especially
the locus Stn90 seems to be a promising candidate to uncover
genes related to adaptive divergence in Fennoscandian
three-spined stickleback populations. Given the availability
of the three-spined stickleback genome sequence, it should
be possible not only to narrow down the genomic regions
2008 The Authors
Journal compilation 2008 Blackwell Publishing Ltd
Acknowledgements
We would like to thank Tuomas Leinonen for collecting the
stickleback samples and Kaisa Vlmki for excellent laboratory
assistance. Anti Vasemgi and two anonymous referees gave
useful comments on an earlier version of this manuscript.
This study was supported by the Finnish Graduate School in
Population Genetics and the Academy of Finland.
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3580 H . S . M K I N E N , J . M . C A N O and J . M E R I L
Appendix 1 Basic population genetic estimates for the loci identified as directional (bold) and balancing (italics) selection outliers in the
global analysis. Allele frequency differentiation (FST), differentiation when taking into account stepwise mutations (RST), pRST is the
permuted FST assuming that stepwise mutations had not contributed to the population differentiation. AR = allelic richness based on 12
individuals, HE = expected heterozygosity, % variation is the amount of variance explained in the locus-by-locus amova analysis between
marine and freshwater populations
Locus
FST
RST
AR
HE
% variation
Stn12
Stn90
Stn365
Stn380
Stn381
Stn122
Stn19
Stn34
Stn57
Stn67
Stn59
Stn81
GAest8
GAest30
GAest36
GAest52
GAest57
GAest60
GAest63
GAest74
0.408
0.521
0.654
0.685
0.856
0.087
0.069
0.065
0.088
0.089
0.040
0.066
0.080
0.047
0.069
0.087
0.087
0.036
0.043
0.061
0.148
0.677
0.727
0.837
0.855
0.0306
0.162
0.167
0.387
0.159
0.180
0.133
0.358
0.462
0.067
0.406
0.067
0.016
0.238
0.239
0.386 (0.140.63)
0.497 (0.140.72)
0.596 (0.280.78)
0.505 (0.080.84)
0.855 (0.850.85)
0.084 (0.00.22)
0.067 (0.00.19)
0.064 (0.00.18)*
0.085 (0.00.25)***
0.088 (0.00.26)
0.040 (0.00.14)**
0.049 (0.00.18)
0.056 (0.00.21)***
0.046 (0.00.17)***
0.068 (0.00.18)
0.083 (0.00.21)***
0.080 (0.00.19)
0.038 (0.00.13)
0.042 (0.00.13)***
0.059 (0.00.16)***
7.7
4.3
2.9
3.3
2.0
13.4
16.5
15.9
15.7
9.7
14.4
13.7
16.0
19.4
11.3
14.3
10.7
11.6
17.3
16.0
0.84
0.72
0.58
0.55
0.49
0.94
0.96
0.95
0.96
0.89
0.94
0.94
0.96
0.98
0.91
0.92
0.89
0.92
0.97
0.95
6.1
14.3
43.5
36.5
51.4
1.1
0.7
1.1
0.4
1.0
0.4
0.1
1.5
0.2
0.9
1.9
0.0
0.7
0.9
0.4
N AT U R A L S E L E C T I O N I N S T I C K L E B A C K S 3581
Appendix 2 A list of the locus names, GeneBank accession numbers and the chromosomal positions in the three-spined stickleback genome. Primer sequences
are given for the markers (GAest), which were developed from the stickleback EST-library available at the National Centre For Biotechnology Information. Note
that the accession number refers to the EST-sequence in case of GAest-loci
Locus
GeneBank Acc.
Chromosome
Stn12
Stn122
Stn19
Stn3
Stn34
Stn57
7033Pbbe*
Stn110
Stn163
Stn21
Stn38
Stn79
1125Pbbe*
Stn132
Stn174
Stn135
Stn195
Stn46
Stn130
Stn26
Stn365
Stn9
Stn96
Stn100
Stn178
Stn185
Stn219
Stn61
Stn82
4147Pbbe*
Stn1
Stn15
Stn23
Stn380
Stn381
Stn70
Stn30
Stn37
Stn52
Stn64
Stn67
Stn59
Stn81
Stn125
Stn90
Stn146
Stn148
Stn119
Stn173
Stn180
Stn170
Stn196
Stn198
Stn205
Stn49
Stn160
Stn208
Stn118
Stn83
GAest1
G72132
G72282
G72135
G72128
G72243
G72155
AJ010360
G72182
G72304
G72136
G72145
G72166
AJ010354
G72193
G72310
G72288
G72221
G72150
G72286
G72240
G72131
G72176
G72177
G72312
G72214
BV102497
G72158
G72168
AJ010358
G72126
G72236
G72137
G72164
G72241
G72144
G72154
G72160
G72161
G72156
G72262
G72189
G72173
G72296
G72198
G72280
G72309
G72313
G72307
G72320
G72222
G72324
G72153
G72229
G72186
G72263
DN712245
I
V
XII
I
VIII
V
XI
IX
XIV
VII
IV
VII
XX
XI
XVI
XII
XVIII
IV
XI
II
IV
I
VIII
IX
XVI
XIX
XXI
VI
VII
IV
I
I
X
IV
IV
VII
III
IV
V
VI
VI
V
VII
X
VIII
XII
XIII
X
XV
Scaffold_182
XV
XVIII
Scaffold_128
Scaffold_27
IV
XIV
XXI
XIV
VIII
IX
GAest3
DN716846
XIX
GAest4
DN736839
XVII
GAest6
DN733971
IV
GAest7
DN735236
VIII
GAest8
DN732699
XXI
GAest11
DN735398
XI
GAest14
DN735932
Primer sequences 53
F: TGAATGCTTCTAATTGGTGTAG
R: AGTCCATGAAAACAAACCTCTA
F: ATTAGAAACCAGATGTCAAAGC
R: TGCGTATACATACATATCACTCAG
F: TAGAAATGAATCAAAACACGAG
R: TGTCAGATGCAAATAAGTGAGT
F: GGTTAACTTCTTTGTCAGCTTC
R: TTAGTTGGATTACAATGTGAGG
F: CTGAAGCAGAAAGTGCTCA
R: TGGTCTATTACTGATGCTCAAA
F: CCTTGGAGGTTTGTTAGTTCT
R: ATCGCAGATAGAGGAATAGAGA
F: TCTCTTACGTTGTATGCACATT
R: TTACACTACTGAAGGACTGCTG
F: CGTTTTATGTGATTCATGGTAG
R: GAACGTACACAAACTGCTACTG
Locus
GeneBank Acc.
Chromosome
Primer sequences 53
GAest15
DN732561
II
GAest16
DN716882
Scaffold_27
GAest17
DN720020
Scaffold_128
GAest19
DN728221
VI
GAest21
DN704336
VII
GAest26
DN700256
GAest29
DN704287
III
GAest30
DN685788
XII
GAest31
DN685475
XIX
GAest32
DN694579
XII
GAest34
DN682722
XXI
GAest35
DN685500
IV
GAest36
DN687963
II
GAest41
DN666064
GAest42
CD494453
VI
GAest43
CD493458
IV
GAest47
DN705394
XIX
GAest49
DN730395
XIV
F: CAATCATGAAACAAGTTACCAG
R: ATCTTTATAAGAGCACACGCTT
F: ATTCAGAAAAGAGAGAGGTGTG
R: ACAGAGTATCCATGCTTCATTC
F: ATTTCACAACATCATCATCATC
R: TATATTCCAGTTTGCAGAAAGA
F: ATGAGAGAGCACATGACTGAG
R: GAAATCAACGGGAACAGATA
F: TATCATTACGGATGACTTCAGA
R: AAGTCCTCATTTCAATGTTTG
F: AAAACACTAAAATGGTCCTTTG
R: ATTTATGGCGTTTATGGATTAG
F: TCCGTCAGTTAGTCTGTTTGTA
R: CTGGACTACTTTACTGTGCTGA
F: AGGTTGGTCTAGTAAAAGCTGA
R: GCCAATCAGGAGAACAACT
F: CAAACTAAGCACAAACTAAGCA
R: GACGTTCATTCATCTCTTCTCT
F: GTAAATATCTCTTGCCAATTC
R: ATATCAATAATGCAGTAGGTTAC
F: ATGACAGACATGAAATGAACAC
R: CAAGTACAAGACGAGCTACGA
F: TGCAGTTTAGCACAAACTCTAC
R: AATGTGTAACCATCACAGAATG
F: CGTAGATCCCAAATAAACTCAT
R: TCATCTCGTCTAATTGTTTCTG
F: TTTACACAAAAGCTTCATAACG
R: AAGGGGTCCAGATAGAATATGT
F: ATTGGCTTGAATAAATGTGG
R: CTCATTAACTGTAGGTGACACG
F: TCTCAGAAAGCAATACAAAA
R: ACTGTTATCACGTCCACTTT
F: CAGCAAAGTTACAGACTGACAT
R: GTGAATTATTTTGTACTGCGAA
F: CTTTAACACCAGTTCATGTCAC
R: CAGATTTGTAAGAAACACATCG
F: GGTTGAATCTGTCTTACCAAAT
R: CAGTATCATGGCTACATCTCAG
F: TACTCATACCAGCTTATGCAAG
R: ACTTGGGTGTTTATAAGTCGTC
F: CAGAAGAAGAACCCCATATTTA
R: GACCCTATCTTCCCATTTATTT
F: CATCAACATCAACAACAACTG
R: GTCAGACAGGGTCTCGTATT
F: TCTTTGTAGTACGATCCAATCA
R: ATTCCATGTCTAATCTCTCAGC
F: GTTAGGATTGAAAAGGAAGGTT
R: TGTACGACTACGACAAGCTG
F: GTTAGCTTCAAAGATCCAAATG
R: AGAATGAGAGCAGTTACAGAGC
F: CTGGAACAACAAACATTTTATC
R: TGGAGGTCTGTTATTTTATTTTC
F: ATGAAAGGATAATGTTACCAGC
R: CAACAAAAGAAATGTGAGAATG
F: AAAGAGATTTTACCTCTGATCG
R: ACGCTTCTTCATACGAGTTAAT
F: GTACTGTGGTTGAGTGTGTGTT
R: CGTGTTTAGATGGAAGTGTAGA
F: AAAAGTGTGAAGTCACTGGAG
R: AGTGTCTACCTCTAACCTACGTG
F: AGGAGGAGTGCAGATAAAGAC
R: ATGAAGATGAAATCAGACGAGT
F: ATGAAAGGATAATGTTACCAGC
R: CAACAAAAGAAATGTGAGAATG
F: CACCACTAACTCGTACATCCTT
R: CTCTATCCATAGTTGTTGCTCC
F: TACAAAGCTCAGTCAGAAGTCA
R: CGACCACAATAACCAGTAGAC
F: CTGATCACTTGTGTGTACTTTGT
R: AAAGAGATCCGAGAGTACGAC
F: AATGTCAAATAACGTCTTCTCC
R: GAAGCCTTTTAACACGTCTAAC
F: GTTTGAATATAGCTTCCTCTGC
R: GTTTTCTTTTGAAACATTCCTC
F: AACACCTCCTTAAACAGATCAC
R: GGGTTAAAAGCAATGAGATTAC
GAest50
DN719394
GAest51
DN728002
XVI
GAest52
DN722519
Scaffold_48
GAest53
DN719509
VII
GAest55
DN698980
III
GAest56
DN683451
XI
GAest57
DN707612
XX
GAest60
CD502157
Scaffold_132
GAest61
DN713857
XII
GAest63
DN693003
XX
GAest64
DN655209
GAest66
DN683768
II
GAes67
DN685529
XIII
GAest71
DN713857
XIII
GAest73
DN730681
GAest74
DN730448
XV
GAest80
DN704459
XI
GAest82
DN706699
VII
GAest84
DN732607
XII
GAest87
DN687948
XVIII
3582 H . S . M K I N E N , J . M . C A N O and J . M E R I L
Appendix 3 Details of the markers, which have been found to be associated with QTLs in experimental crosses and were used in this study
to identify traces of natural selection in the morphological traits. Note that group refers to the linkage groups in the original linkage map
(Peichel et al. 2001)
Markers
Group
Traits
LOD
% explained
Reference
Stn178
Stn9
Stn26
Stn96
Stn130
Stn82
XVI
I
II
VIII
XI
VII
IV
Stn21
Stn365
Stn380
Stn381
1125Pbbe
II
IV
IV
IV
XXV
Stn219
Stn185
XXVI
IX
37
21
17
22
17
19.8
3745
3745
3745
4.9
116.9
4.7
4.9
NA
NA
NA
26.3
10.9
3.512.3
7.8
14.9
1.53
1.42
2.72
2.57
22.2
43.7
c. 40
c. 40
5.8
77.6
5.6
7.6
NA
NA
NA
28.9
17.9
4.528.6
32
Gac4174
# rakers
Dorsal Spine1
Dorsal Spine1
Dorsal Spine2
Dorsal Spine2
Asc. Pelvic Branch
Pelvic Spine
Pelvis Loss
Pelvic Asymmetry
Pelvic Spine
Lat. Plates
Pelvic Girdle (length)
Pelvic Spine
# lateral plates
# lateral plates
# lateral plates
Plate Width
Plate high
# lateral plates
Opercular bone shape
Appendix 4 Allele frequencies in the microsatellite locus Stn365 showing low within-population heterozygosities but skew between
population allele frequencies. This pattern might explain the differences in detecting selection with FST and gene diversity ratio based
neutrality tests
Allele (bp)
Merirastila
Orrevatnet
Barents
L. Pulmanki
L. Kevo
L. Vttern
R. Neretva
138
140
142
HE
10.5
85.5
4.0
0.26
4.2
89.6
6.3
0.19
98.0
2.0
73.0
25.0
0.41
87.0
13.0
100
71.7
28.3
0.40
2.0
0.04
0.23