Contrasting patterns of genetic variation in core and peripheral populations of highly outcrossing and wind pollinated forest tree species
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Contrasting patterns of genetic variation in core and peripheral populations of highly outcrossing and wind pollinated forest tree species
Bła z_ej Wojkiewicz 1
Monika Litkowiec 1
Witold Wachowiak 0 1
Associate Editor: Kristina Hufford
0 Faculty of Biology, Adam Mickiewicz University, Institute of Environmental Biology , Umultowska 89, Poznan 61-614 , Poland
1 Institute of Dendrology, Polish Academy of Sciences , Parkowa 5, Kornik 62-035 , Poland
Gene flow tends to have a homogenising effect on a species' background genetic variation over large geographical areas. However, it is usually unknown to what extent the genetic structure of populations is influenced by gene exchange between core and peripheral populations that may represent stands of different evolutionary and demographic history. In this study, we looked at the patterns of population differentiation in Scots pine-a highly outcrossing and wind pollinated conifer species that forms large ecosystems of great ecological and economic importance in Europe and Asia. A set of 13 polymorphic nuclear microsatellite loci was analysed to infer the genetic relationships among 24 populations (676 individuals) from Europe and Asia Minor. The study included specimens from the primary continuous range and from isolated, marginal stands that are considered to be autochthonous populations representative of the species' putative refugial areas. Despite their presumably different histories, a similar level of genetic variation and no evidence of a population bottleneck was found across the populations. Differentiation among populations was relatively low (average FST ¼ 0.035); however, the population structure was not homogenous, which was clearly evident from the allelic frequency spectra and Bayesian assignment analysis. Significant differentiation over short geographical distances was observed between isolated populations within the Iberian and Anatolian Peninsulas (Asia Minor), which contrasted with the absence of genetic differentiation observed between distant populations e.g., between central and northern Europe. The analysed populations were assigned to several groups that corresponded to the geographical regions of their occurrence. These results will be useful in genetics studies in Scots pine that aim to link nucleotide and phenotypic variation across the species distribution range and for development of sustainable breeding and management programs.
Demographic history; genetic structure; glacial refugia; phylogeography; Pinus sylvestris; population history; recolonization
VC The Authors 2016. Published by Oxford University Press on behalf of the Annals of Botany Company.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/
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Demographic and evolutionary processes interplay to
shape genetic variation that is crucial to maintaining
species’ adaptive responses to changing environments.
Genetic variation among plant populations in the
Northern Hemisphere has been shaped by range shifts
and recolonization following the last glacial maximum
(25–18 000 years ago)
(Petit et al. 2003; Nosil and Feder
. The assessment of the genetic relationships
among natural populations across the species distribution
is important for tree management and for breeding and
gene conservation programs, particularly in the face of
ongoing environmental changes (Savolainen et al. 2011).
Forest trees are known to form large, wind pollinated
populations and maintain a high level of genetic and
phenotypic variation in comparison to other plant
(Petit and Hampe 2006)
. High diversity ensures that
these long-lived organisms can survive and evolve under
changing environmental conditions. Studies of historical
processes such as population size fluctuations and
geographical range shifts are needed to better understand
the effect of demographic factors that influence
background genetic variation and to effectively contrast
neutral variation with that resulting from natural selection
(Luikart et al. 2003; Li et al. 2012)
. However, currently
available genetic data still lack sufficient information to
fully reflect the usually complex demographic history of
many forest tree species. For highly outcrossing and
wind pollinated species, gene flow is supposed to have
homogenising effects on the background, neutral
genetic variation between populations over large
geographical distances. However, its effect on the
distribution of genetic variation between populations
from core range vs. marginal populations of the species
is not that clear, especially for long-lived temperate
forest tree species.
In the present study, we focused on Scots pine (Pinus
sylvestris), which is one of the most ecologically and
economically important forest-forming tree species in
Eurasia. Some former phylogeographic studies of this
pine based on isozyme polymorphisms, organelle DNA
and palynological records have shown that the most
abundant populations of Scots pine survived the cold
periods of the Pleistocene within southern Eurasia in the
Iberian, Apennine and Balkan Peninsulas and in the
Anatolian and Caucasus Mountains
(Willis et al. 1998;
Willis and van Andel 2004; Cheddadi et al. 2006;
Naydenov et al. 2007; Pyha€ja€rvi et al. 2008; Soto et al.
2010; Buchovska et al. 2013)
. The Alps, the Carpathians
and Moscow are considered refugial areas for other cold
tolerant conifer species including Norway spruce (Picea
(Tollefsrud et al. 2008, 2009)
. Although there are
strong indications regarding the location of some
putative refugial stands, it is less clear how migration and
gene flow have influenced the patterns of genetic
variation at neutral gene markers across the present range of
the species. Consequently, the relationships among the
gene pools of populations from core and peripheral
distribution are not well elucidated.
The aim of this study was to assess the genetic
variation and structure of Scots pine populations from central
and north Europe in relation to its marginal populations
from the European and south-west Asiatic refugial areas
based on the analysis of nuclear simple sequence repeat
(nSSR) markers. Nuclear markers in pines are distributed
by seeds and pollen and could potentially be dispersed
at large geographical distances. Therefore, the
identification of populations with divergent genetic backgrounds
could suggest the existence of distinct populations that
do not share a recent history. To our knowledge, this
species has not yet been investigated on such a large
geographical scale using this type of neutral marker. In this
study, we used a set of 13 nSSR loci to examine genetic
diversity and test for the existence of populations of
different origin. We aimed to check if there is any difference
between core and peripheral populations considering
the presumably homogenising effect of long distance
gene flow. Information regarding the genetic
relationships among populations is particularly important to
advance studies of the genetic architecture of observed
variation in phenotypic traits that have been shaped by
selection and local adaptation within the Scots pine
Plant material, DNA extraction and microsatellite genotyping
The study comprised 24 Scots pine populations (Fig. 1), 8
of which were from the core continuous species
distribution from central and northern Europe. The rest of the
populations analysed were collected from isolated,
peripheral stands on the Iberian and Anatolian Peninsulas,
the Massif Central of France, Scotland and the
Balkans [see Supporting Information—Table S1]. The
number of samples per population varied from 22 to 49
individuals with a total of 676 individuals (Table 1).
Genomic DNA was extracted from needles using a
(Dumolin et al. 1995)
. The initial set of 22
nuclear microsatellite markers originally identified in pine
(Soranzo et al. 1998; Elsik et al. 2000;
Liewlaksaneeyanawin et al. 2004; Sebastiani et al. 2012)
were screened for their ability to provide repeatable, high
quality results, sufficient polymorphism and unambiguous
VC The Authors 2016
allele binding. The final set of loci used in this study
included 13 nSSRs that provided high-quality amplification
products. DNA amplification was carried out in three
multiplex reactions including loci psyl2, psyl18, psyl25, psyl36,
psyl42, psyl44 and psyl57 (multiplex 1); Spag7.14,
PtTX2146, PtTX3107 and Spac11.4 (multiplex 2) and
PtTX3025 and PtTX4011 (multiplex 3). The PCR for each
sample was conducted in a total volume of 10 lL
containing 5 lL of Qiagen Multiplex Master Mix (2 ), 0.2 lL of
primer mix (20 lM), 1 mL of Q-Solution (5 ), 0.8 lL
RNasefree water and 3 lL of DNA 84 template (approximately
10–20 ng). The following PCR amplification conditions
were used: multiplex 1, initial denaturation at 95 C for
15 min, 38 cycles of denaturation at 94 C for 30 s,
annealing at 57 C for 90 s, extension at 72 C for 90 s and final
extension at 72 C for 10 min; multiplex 2, initial
denaturation at 95 C for 15 min, 30 cycles of denaturation at 94
C for 30 s, annealing at 55 C for 90 s, extension at 72 C
for 90 s and final extension at 72 C for 10 min; multiplex
3, initial denaturation at 95 C for 15 min, 10 cycles of
denaturation at 94 C for 30 s, annealing at 60 C for 40 s
with temperature decreasing by 1 degree per cycle,
extension at 72 C for 90 s, 36 cycles of denaturation at 94 C
for 30 s, annealing at 50 C for 40 s, extension at 72 C for
90 s and final extension at 72 C for 10 min. The
fluorescently labelled PCR products, along with a size standard
(GeneScan 500 LIZ), were separated on a capillary
sequencer ABI 3130 (Thermo Fisher Scientific, Waltham,
USA). The allele’s size was determined using GeneMapper
software (ver. 4.0; Life Technologies).
Allelic diversity and within-population genetic variation
Genotypic disequilibrium between pairs of loci was
tested at the single population level and across all
populations with a Fisher’s exact test using ARLEQUIN 3.11
(Excoffier and Lischer 2010)
. The allelic diversity of the
studied loci and within-population genetic variation were
estimated based on the following parameters: the
number of alleles per locus (Al), the mean number of alleles
per population (Np), the mean number of effective alleles
per population (Ne), the mean number of private alleles
per population (Pa), the observed heterozygosity (Ho)
and the unbiased expected heterozygosity (He), all of
which were computed using GenAlEx 6
. In accordance with earlier studies that
showed that microsatellites are known to be susceptible
to genotyping errors
(Guichoux et al. 2011)
, the null allele
frequency for each loci was calculated using the EM
algorithm with FreeNA software
(Chapuis and Estoup 2007)
We used FSTAT v 2.9.3
to estimate gene
VC The Authors 2016
diversity (Gd), rarefied allelic richness (AR22) for a
minimum sample size of 22 individuals and inbreeding
coefficient values (Fis). The deviation of genotypic frequencies
from Hardy–Weinberg equilibrium (HWE) were also
identified utilizing the inbreeding coefficients
(Fis; Weir and
with a correction for null alleles (FisNull)
for each population using the Bayesian method
implemented in INEST 2.0 software
evaluation was performed using the IIM model with 100 000
MCMC iterations, storing every 100th value and with a
burn-in period of 10 000. A Bayesian procedure based on
the Deviance Information Criterion (DIC) was used to
determine the statistical significance of the inbreeding
component by comparing the full model with the
random mating model (under the assumption Fis ¼ 0).
Genetic differentiation between populations
To estimate the proportion of the total genetic variation
due to differentiation among populations, an Analysis of
Molecular Variance (AMOVA) based on two distance
methods (FST and RST) was conducted using ARLEQUIN
3.11. Moreover, due to the presence of null alleles, the
global, pairwise and within-geographic regions FST were
calculated using FreeNA software. FreeNA applies the
ENA (Excluding Null Alleles, FSTENA) correction method
to effectively correct for the positive bias induced by the
presence of null alleles in the FST estimation. Bootstrap
95 % confidence intervals (CI) were calculated for the
global FSTENA values using 2000 replicates across the
loci. The statistical significance of the FST values was
verified with ARLEQUIN 3.11.
To evaluate the ability of the stepwise mutation model
(SMM) to differentiate among populations and
geographical regions, which in turn indicates whether
phylogeographical structures exist, the computed FST and RST
were compared. To test whether the difference between
values of RST and permuted pRST (which corresponds to
FST) was significant, the permutation test proposed by
Hardy et al. (2003)
was implemented in the program
(Hardy and Vekemans 2002)
The genetic population structure (in the case of
microsatellite markers) can arise due to isolation by distance
(IBD), range expansions, diffusion of genes through space
in migratory events and/or allelic surfing
(Diniz-Filho et al.
. Because of that a
Mantel test (1967
) was applied to
evaluate spatial processes driving populations structure by
comparing the matrixes of pairwise geographic
(logarithmic scale) and pairwise genetic (measured as FST/(1 FST))
distances. The statistical significance of the correlation
was calculated for all populations and sets of populations
located along latitudinal and longitudinal transects using
9999 permutations with GenAlEx 6.
Principal Coordinates Analysis (PCoA) was applied to
visualize the patterns of the genetic structure of the
populations using a pairwise FSTENA matrix and GenAlEx 6
software. Phylogenetic relationships between the
populations were investigated using POPTREEW
et al. 2014)
. The phylogenetic tree was constructed from
allele frequency data using the neighbour-joining (NJ)
method. This method allows faithful depiction of genetic
structure for some populations that have an
isolationby-distance population structure
standard genetic distance (DST)
as a distance measure for the construction of the
phylogeny. The statistical robustness of the branches was
evaluated with 1000 bootstrap replicates.
The assignment of individuals and populations to
genetically distinct groups was conducted using the Bayesian
clustering method with the software STRUCTURE 2.3.4
(Pritchard et al. 2000; Falush et al. 2003; Hubisz et al.
. This program uses a Markov chain Monte Carlo
(MCMC) algorithm to assign individuals to a given number
of genetic clusters (K) without considering sampling
origins and assuming that each cluster is in optimal
Hardy–Weinberg (H–W) and linkage equilibrium (LE). The
correlated allele frequencies and admixture model used
allowed for mixed recent ancestry of individuals and
assigned the proportion of the genome of each individual to
the inferred clusters. Moreover, because all the
microsatellite loci used in this study were affected by null alleles (see
Results section), the recessive alleles option was chosen.
Twenty independent runs were performed for each K,
from K ¼ 1 to 24, with burn-in lengths of 500 000 and 100
000 iterations. The probability distributions of the data
(LnP[D]) and the DK values
(Evanno et al. 2005)
visualized in the STRUCTURE HARVESTER Web application
(Earl and von Holdt 2011)
. Following Bayesian clustering,
the hierarchical distribution of genetic variation was
characterized using an analysis of molecular variance
(AMOVA). A three-level AMOVA was conducted in
ARLEQUIN 3.11 and significance was obtained via 10 000
Tests for genetic bottleneck model
The program Bottleneck v.1.2.02
(Cornuet and Luikart
was used to evaluate whether the examined
populations suffered a severe genetic bottleneck or had
experienced recent reductions in their size. A
Wilcoxonsigned rank test of heterozygosity excess was used to
evaluate the significance of a potential bottleneck. The
analysis was performed under three different models of
microsatellite evolution: the SMM model, the infinite
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allele model (IAM) and the two-phases model of
mutation (TPM) with parameters of 30 % multiple-step
mutations and 70 % single-step mutations. In addition, the
distribution of allele frequencies over all loci was
examined for a ‘mode-shift’, which might indicate a
bottlenecked population rather than a stable population.
Allelic diversity and within-population genetic variation
The 13 nuclear microsatellite loci investigated were
polymorphic, providing a total of 160 size variants. There was
no significant linkage disequilibrium between pairs of
loci across all populations (P > 0.01). The number of
alleles per locus ranged from 3 (psyl25) to 40 (Spag7.14),
with an average of 12.3 [see Supporting Information—
Table S2]. The estimated frequency of null alleles for
most of the loci was moderate (< 6 %, but
generally < 2 %) with the exceptions occurring at three loci:
psyl18 (8.7 %), Spag7.14 (8.3 %) and PtTX3107 (16.2 %)
[see Supporting Information—Table S2].
The basic statistics for genetic variation within the
populations are summarized in Table 1. More than five
alleles were observed in each population with an
average Ap ¼ 6.08. The allelic richness measures obtained
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based on a minimum of 22 samples (AR22) were
comparable in all investigated populations and ranged from 5.1
in the T1 population to 6.2 in the PL1 population. The
mean effective number of alleles was Ae ¼ 3.4. The
lowest number of effective alleles was observed in the
Tokat–Yıldızeli population from Turkey (T3, Ae ¼ 2.9),
whereas the highest number of effective alleles was
found in the population from southwestern Poland (PL1,
Ae ¼ 3.8). Twenty private alleles were also detected
among some of the studied populations, and their
frequency ranged from Pa ¼ 0.0 % to Pa ¼ 23.1%. Similar
levels of gene diversity were found in all populations,
which ranged from 0.48 to 0.53. However, pine
populations from the peripheral stands appear to show greater
diversity (with an average of 0.52) in comparison with
populations from central and northern Europe (with an
average of 0.48). The level of the overall observed
heterozygosity (Ho) per population (average ¼ 0.45, range:
0.38–0.49) was similar for all populations and slightly
lower than the level of expected heterozygosity (He)
(average ¼ 0.50, range: 0.44–0.54). The inbreeding
coefficients ranged from 0.017 in the F2 population from
Finland to 0.282 in the T5 population from Turkey, with
an overall mean of 0.120. However, the inbreeding
coefficients may be highly overvalued due to the presence of
null alleles. Because of this and based on our previous
results that showed that null alleles were present at all
investigated loci [see Supporting Information—Table
S2], we used the IIM approach (see M&M) to partition out
their influence on FisNull (inbreeding coefficients with null
alleles correction) value. Recalculated values of
inbreeding coefficients, taking into account the frequency of null
alleles (FisNull), were much lower than those obtained
previously and ranged from 0.014 to 0.081 with an average
of 0.045. The values of FisNull and Fis were significantly
different from zero in all populations. However, it seems
that deficiency of heterozygotes is mostly due to the
presence of null alleles. Moreover, it should be noted that
the highest values of both Fis and FisNull were observed in
peripheral pine populations from Turkey and Spain.
Genetic differentiation between populations
The analysis of molecular variance (AMOVA) based on
the number of different alleles (FST) and the sum of the
squared size differences (RST) showed that differentiation
between Scots pine populations was low but significant
(FST ¼ 0.035, RST ¼ 0.032; P< 0.0001). The majority of the
variance was found within populations (Table 2). The
global FST values, estimated both with and without the
ENA correction, was FST ¼ 0.035 and FSTENA ¼ 0.037,
respectively. The similarity of these values implies that the
presence of null alleles is not a significant factor
affecting the level of genetic differentiation. The results
of genetic differentiation among populations within the
studied geographical regions are presented in Table 2.
The greatest differentiation was found between
populations from Turkey and Spain, whereas the within-region
FST values obtained for the Balkans, Poland and Finland
were not statistically significant. Most of the pairwise FST
population values were significant (P < 0.001) [see
Supporting Information—Table S3]. The greatest differ
ence (0.11) was between the T4 population from C¸atacık
in Turkey and the H2 population from the Sierra de Neila
in Spain, and the lowest (FST < 0.01) was between the
PL2 population located in southern Poland and the PL4
population located in northern Poland.
The permutation test, by which global RST was
compared against the distribution of 10 000 pRST values, did
not detect a significant difference between these
parameters (RST ¼ 0.032; pRST ¼ 0.025; CIpRST 95 % ¼ 0.01–0.04;
pH1: RST > pRST ¼ 0.164). This suggests an absence of
phylogeographic structure and that gene flow is high
compared with the mutation rate. However, the results of the
Mantel test correlation between genetic distance and
the logarithm of geographic distance among all P.
sylvestris populations indicated that the genetic diversity is
structured in geographic space (R ¼ 0.39, P < 0.05). The
strongest correlation was found along a longitudinal
transect among populations from Anatolia and the
Iberian Peninsula (R ¼ 0.73, P < 0.05). The spatial genetic
structure among all populations was rather weak,
whereas only 15 % (R2 ¼ 0.152) of the genetic divergence
was explained by geographical distance. The strong
structure (53 % of the genetic divergence could be
explained by geographical distance) found among south
peripheral populations is most likely due to the presence
of geographical barriers along Mediterranean basin
transects which intensify the effects of the process of
isolation by distance (IBD).
Population clustering and phylogenetic relationships
The genetic structure of the Scots pine populations
based on the pairwise FSTENA matrix is illustrated in the
PCoA plot (Fig. 2). The east–west subdivision of the
southernmost peripheral populations was clearly shown
by the first coordinate, which explained more than 25 %
of the variation. The second variable (second coordinate),
which was responsible for more than 20 % of the total
variation, separated the pine populations from central
and northern Europe from those in southern Eurasia.
Moreover, according to the FST values obtained for each
geographical region (Table 2), populations from both
Turkey and Spain formed a much more heterogeneous
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Source of variation d.f. Sum of squares Variance components Percentage of variation P
group in comparison with populations from the Balkans
and central and northern Europe.
The relationships between populations are illustrated
in Supporting Information—Figure S1, which shows the
phylogenetic tree based on the NJ method [see
Supporting Information—Figure S1].
The Bayesian assignment of samples obtained with
STRUCTURE indicated that three clusters (K ¼ 3) provide
the most probably representation of the overall genetic
structure of the analysed Scots pine populations. The
admixture proportions of each of the three gene pools
(clusters) were estimated and differed among
populations (Fig. 3). The highest frequency of cluster 1
(indicated in red) was found in populations from Turkey
(T1, T2, T3, T4 and T5), with an average of 77.6 %,
whereas cluster 2 (indicated in green) was most
frequent in populations from Poland and Finland (PL1,
PL2, PL3, PL4, F1, F2, F3 and F4), with a mean of
61.5%. Three populations from Spain (H1, H2 and H3)
showed the highest frequencies of cluster 3 (indicated
in blue; mean value of 69.1%). Pines from Ukraine,
Balkans, north of Spain, Massif Central in France and
Scotland were classified as mixed populations.
However, the gene pool of the population from
Ukraine exhibited a predominance of cluster 1,
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whereas populations from the Balkans showed a
predominance of cluster 2. Gene pools from north of the
Iberian Peninsula (A, H4) and the population from
France exhibited a higher frequency of cluster 3.
Clusters 2 and 3 contained the dominant frequencies
in the gene pool of Scottish pine populations, with a
higher frequency of cluster 2.
Based on these findings and the pairwise FST matrix
[see Supporting Information—Table S3], an AMOVA
analysis was conducted between six groups of
populations. The largest group consisted of the populations
from Turkey and Ukraine (Group 1). The next largest
groups included populations from the Balkans (Group
2), southern Spain (Group 3), north of the Iberian
Peninsula (populations H4 and A) and the population
from the Massif Central in France (Group 4). Two other
groups were formed by the population from Scotland
(Group 5) and the populations from central and
northern Europe (Group 6). The AMOVA results and the
pairwise FST values between these groups of populations
are presented in Tables 2 and 3, respectively.
Significant genetic differentiation over short
geographical distances was observed between the populations
within Spain and Turkey (Table 2) but not between
populations from the Balkans, Poland or Finland. The
highest degree of genetic differentiation was found
between southern Spain (Group 3) and Turkey and
Ukraine (Group 1) and between those geographical
areas and the rest of the populations (Table 3; see
Supporting Information—Table S3).
Tests for genetic bottleneck model
Despite the fact that some of the investigated
populations were characterized by lower genetic diversity, no
evidence for a recent genetic bottleneck or reductions
in effective population size under the two-phased
mutation model (TPM), infinite allele mutation model
(IAM) or stepwise mutation model (SMM) was detected.
Moreover, the mode-shift test, based on the frequency
distribution of alleles, demonstrated a typical L-shaped
mode for all populations, which is consistent with a
Genetic variation and differentiation
In our study, we evaluated the neutral genetic variation
and genetic structure of Scots pine populations from the
core and peripheral distribution in Europe and Asia Minor
based on variation in nSSR markers. All the 13
microsatellite loci studied were polymorphic with a mean of 12.3
alleles per locus. Considerably lower numbers of alleles
were found at the psyl loci (an average of 6.1) compared
with the Spag and PtTX loci (an average of 30.5 and 14.0,
respectively). The most variable loci were Spag7.14,
Spac11.4 and PtTX2146. Therefore, these loci appear to
be the most informative for population genetics studies
of P. sylvestris that require a high resolution of markers
for fine-scale analysis. Consistent with earlier studies on
the genetic variation of conifer species, we found that
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the analysed loci contained some level of null alleles
(Liewlaksaneeyanawin et al. 2004; Belletti et al. 2012;
Sebastiani et al. 2012)
. However, as evidenced by our
dataset, they did not significantly affect the observed
patterns of variation across the studied populations.
Our data show that most of the genetic variation is
located within the Scots pine populations. The level of
genetic variation, as measured by basic statistics, was high
and very similar across populations (Table 1) which is
typical to woody species
(Hamrick et al. 1992)
. The levels
of observed heterozygosity were only slightly less than
the values of unbiased expected heterozygosity and
were primarily due to the presence of null alleles. These
findings indicate that there is no significant allele
frequency disequilibrium in any of the studied Scots pine
populations. There were, however, some noticeable
differences in the parameters describing the
withinpopulation genetic variation of peripheral populations vs.
populations from the core distribution. In general, the
peripheral populations had higher frequencies of private
alleles, with the exception of the Balkan populations. An
excess of private alleles was also observed in the
peripheral Scots pine populations from Spain and Italy based
on an analysis of allozyme and nSSR loci
(Belletti et al.
2012; Prus-Głowacki et al. 2012)
. Those populations had
also higher inbreeding coefficients in comparison with
populations from the Balkans, Poland and Finland. The
results of this study indicate isolation and probably a
limited effective population size in the peripheral
populations from Turkey (T2, T3, T4 and T5) and the south of
Spain (H1, H2 and H3). The stronger isolation of these
pine populations from the south regions apparently
restricts gene flow and seems to cause a more distinct
pattern of geographical variation in these studied regions.
This conclusion is supported by the results of the Mantel
test correlation analysis, which is based on genetic and
geographical distance. We found that there was strong
spatial structure and overall among-population
differentiation between southern peripheral populations. This
high differentiation most likely results from action of the
processes of isolation-by-distance and genetic drift,
which are most effective in isolated stands. It must be
pointed, however, that the isolation did not have a strong
negative impact on the level of within population genetic
variation found in the peripheral pine populations.
Similarly relationship was already demonstrated in the
case of other plant species living in isolation, as for
example in the Cheddar pink (Putz et al. 2015). Our results
also show contrasting patterns of genetic variation
between populations located within distinct geographical
areas. Populations within the Iberian and Anatolian
Peninsulas are separated by relatively short geographical
distances but showed much higher divergence
compared with populations from northern Europe and
the Balkans, which are separated by several thousand
kilometres. In some previous studies, relatively high
genetic diversity (FST ¼ 0.058) was found within the Scots
pine natural range in Italy
(Belletti et al. 2012)
compared to differentiation between populations from the
(FST ¼ 0.02, Karhu et al.1996)
Similarly, a high level of differentiation between P.
sylvestris populations from mountain regions in Spain in
relation to other European populations has been found
(Prus-Głowacki and Stephan 1993; Prus-Głowacki et al.
. Moreover, a high level of differentiation between
populations of Norway spruce from Bulgarian mountains
in comparison to populations from other regions of
natural distribution of this species in Europe was noted by
Tollefsrud et al. (2008)
. Mountain regions of southern
Europe are assumed to have provided particularly
suitable habitats for species survival during last glacial
period, because they allowed species to respond to climate
oscillations using the altitudinal gradient
Our results support the hypothesis of a possible
longlasting isolation of pinewoods in separate Iberian refugia
(Rubiales et al. 2010)
and some pine populations in
separate refugial areas within the Anatolian Peninsula.
Moreover these findings suggest that the homogenizing
effect of gene flow via pollen dispersal on gene pool
variation across geographical ranges may be limited.
The genetic relationships between populations
Although only a small percentage of the total variation
was due to differentiation among populations
(approximately 4%), we found clear signals of population
substructuring at nSSR loci within the Scots pine distribution.
Based on the analysis of allelic frequency spectra and
population assignment methods, we could distinguish
several groups of populations from distinct geographical
locations. Five groups of populations are clearly
represented at the PCoA plot where Scottish population is
grouped with the populations from north of Iberia
Peninsula and Massif Central in France. However taking
into account the admixture proportions of each of the
three gene pools in the analysed populations and the
result of phylogenic relationship analysis, we decided to
separate the population from Scotland as a distinct
group because it could not be clearly attributed to any of
the designated previously groups of populations. As a
result, the AMOVA analysis was conducted between six
groups of populations. The east–west subdivision was
clearly shown between southernmost groups of
populations from Turkey (Group 1) and Spain (Group 3). The
differentiation between the Iberian and Anatolian
populations was also the greatest based on the
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morphological and anatomical traits of needles from
Scots pines in the Mediterranean Basin
(Jasinska et al.
. Phenotypic and genetic differentiation among
populations from these regions support the isolation of
P. sylvestris in the southern European and Anatolian
mountain regions and suggest that these populations
are characterized by a different population history. In
our study, the populations from Turkey and the south of
Spain were significantly different from populations in the
Balkans (Group 2), north of the Iberian Peninsula and the
Massif Central (Group 4), Scotland (Group 5) and Poland
and Finland (Group 6). In some previous mitochondrial
DNA studies, unique haplotypes not observed in other
parts of Europe were detected in Spanish and Turkish
(Cheddadi et al. 2006; Naydenov et al. 2007;
Pyha€ja€rvi et al. 2008)
. Our data show a divergence
between populations within the Iberian Peninsula and
indicate that southern populations have distinct gene pools
compared with northern Spain and Andora populations.
The latter population has probably contributed to the
recolonization of Europe (or was admixed) after the last
glacial maximum, as evidenced by genetic variation
similar to populations from Massif Central and the Balkans
and minor differentiation from populations within the
continuous distribution. The subdivision of populations
from the Iberian Peninsula has also been suggested by
the analysis of biochemical and molecular markers
(Prus-Głowacki et al. 2003, 2012; Robledo-Arnuncio et al.
The position of the Scottish populations is not
straightforward. Scottish populations showed some
similarity to groups from northern Spain and the Massif
Central and populations from central and northern
Europe. Previous pollen, allozyme and monoterpene
studies suggested a west/east population subdivision
within Scotland, reflecting different origins of the
populations contributing to the postglacial colonization
(Birks 1989; Kinloch et al. 1986; Sinclair et al. 1998)
Based on this finding, two hypotheses have been put
forth to explain this phenomenon.
Kinloch et al. (1986)
suggested that the western Scotland pine populations
are relicts and might have survived the last glacial
maximum in some region of the British Isles because
they show relatively little genetic affinity to pines from
the continuous range. The eastern populations were
likely admixed with populations of continental origin
or derived from that distribution. Scottish populations
also show high levels of genetic polymorphism at
nuclear gene loci and patterns of allelic frequency
incompatible with a simple model of population expansion
from mainland locations
(Wachowiak et al. 2011)
Given predictions regarding the extent of the British ice
sheet during the last glaciation
(Clark et al. 2012)
the geographic distribution of some unique
mitochondrial haplotypes, it appears more probable that
colonization of the Scottish Highlands occurred from at least
two different sources
(Sinclair et al. 1998; Soranzo
et al. 2000)
. Our data show that the composition of the
gene pool of the Scottish population has
characteristics in common with populations from both central
Europe and north of the Iberian Peninsula. We also
found high frequencies of private alleles in this
population. However, our data are too limited to provide any
definitive answer regarding the source of the probable
Scots pine admixture in the Scottish Highlands.
The Scots pine populations from the continuous range
in Poland and Finland were characterized by the most
homogeneous gene pools, and differentiation between
populations from those regions was not statistically
significant. The low level of genetic differentiation between
Scots pine populations from central and northern Europe
was also observed for isozymes
(Prus-Głowacki et al.
2012; Sannikov and Petrova 2012)
diversity at nuclear gene loci (Wachowiak et al. 2014). These
results imply free gene exchange among those
populations and that they probably share a common postglacial
history. Moreover, our data show that populations from
the Balkans share gene pools with populations from
central and northern Europe. More challenging is the
identification of putative source populations for the
recolonization of pines following the last glacial
maximum. None of the analysed populations in our study
showed exceptionally high genetic variation or evidence
for recent bottlenecks following the patterns of
nucleotide sequence variation at nuclear gene loci
et al. 2007; Wachowiak et al. 2009; Kujala and
. Taking into account findings about
genetic consequences of glacial isolation and postglacial
colonization on the genetic structure of other cold
tolerant tree species e.g. silver birch (Betula pendula) and
Norway spruce (Picea abies) which at present co-occur
with Scots pine across large parts of its natural habitat,
we can conclude that Europe most likely was
reoccupied by at least three main waves of recolonization.
The source populations could have their origins in the
regions of Alps, Balkans and east refugia with origin at
intermediate latitude in Moscow region
(Palme´ et al. 2003;
Willis and van Andel 2004; Maliouchenko et al. 2007;
Tollefsrud et al. 2008; Parducci et al. 2012)
. These several
distinct lineages of colonization might have mixed in
central Europe as suggested based on colonization
routes of Norway spruce
(Dering and Lewandowski
. However, according to results obtained by
Parducci et al. (2012)
which suggest that conifer trees
might have also survived the last glaciation in the
icefree refugia of Scandinavia, it appears that detailed
VC The Authors 2016
studies of populations from Scandinavia and the eastern
distribution in Asia, including regions of Moscow
(Buchovska et al. 2013)
, are needed to test the possible
recolonization trajectories of the species to the central
Our data show that isolated populations from southern
Eurasia show a high genetic divergence over a short
geographical distance, which contrasts with the
pattern of variation in populations from the northern part
of Europe. A clear subdivision was found between
populations from distinct parts of the Eurasian distribution.
With the exception of the southernmost stands,
populations from the central and northern parts of the
studied distribution range showed some genetic similarity
that may reflect their shared postglacial history or
effective admixture between populations of different
origin. High-resolution mtDNA markers dispersed by
seeds across small geographic areas in pines would be
needed to verify migration trajectories and the location
of source populations for the species’ postglacial
Sources of Funding
This work was financially supported by the Polish
National Science Centre
Contributions by the Authors
W.W. and B.W. designed and conceptualized the study.
B.W. and M.L. collected and analysed the data. B.W.
wrote the manuscript; W.W. assisted in drafting the
manuscript; B.W. and W.W. critically reviewed and
revised the manuscript for content; all authors read and
approved the final manuscript.
Conflict of Interest Statement
We thank Krystyna Boratynska and Jacek Oleksyn for
providing some of the plant materials used in the study.
The authors would like to express their thanks to
Weronika Z_ukowska, the editor and two anonymous
reviewers for constructive comments on the previous
version of the article.
The following additional information is available in the
online version of this article —
Table S1. Populations of Pinus sylvestris used in this
Table S2. Descriptive statistics for the thirteen nuclear
microsatellite loci used in this study. Al, number of
alleles; ANullFreq, mean frequency of null alleles.
Table S3. Pairwise FSTENA matrix for 24 Scots pine
populations (*p < 0.001).
Figure S1. Phylogenetic tree of 24 Scots pine
populations based on Nei’s standard genetic distance (DST)
at 13 nSSR loci (1000 bootstraps) using the
neighbourjoining method (NJ).
VC The Authors 2016
VC The Authors 2016
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